Catherinel Notebook1

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cadoyle, 25 August 2013

I meet with Dr. Campbell on Wednesday, 21st of August 2013. We discussed finishing up my thesis proposal by adding in the aptamer sequences for caffeine and 3-methylxanthine that I will be designing riboswitches for.

I found caffeine's aptamer from Alpha Diagnostic Intl. Inc [[1]]. I downloaded the paper: File:Ferguson et al. 2004.pdf that characterized the aptamer and obtained the sequence. On Alpha Diagnostic Int. Inc website there is a product data sheet for the caffeine aptamer that will be useful [[2]].

I found the apatmer for 3-methylxanthine from File:Soukop et al. (2000).pdf. I found this paper while I was doing research for my proposal. I read in File:Lee et al. (2010).pdf that the 3-methylxanthien aptamer had been discovered from a mutation in the theophylline aptamer. Lee et al. (2010) cited Soukop et al. (2000) for the characterization of the 3-methylxanthine aptamer. In Soukop et al. (2000) they list two aptamers for 3-methylxanthine. I picked the aptamer with C22 mutation only because it had a stronger affinity for 3-methlyxanthine (Figure 2C) despite Figure 1B showing that it's specificity for 3-methylxanthine is low.

Name	Sequence	Reference
Caffeine	5-GGAUGUCCAGUCGCUUGCAAUGCCCUUUUAGACCCUGAUGAGGAUCAUCGGACUUUGUCCUGUGGAGUAAGAUCG CGAAACGGUGAAAGCCGUAGGUCU-3	Ferguson et al. (2004)
3-methlyxanthine	5- AUACCAGCCGAAAGGCCAUUGGCAG-3	Soukop et al. (2000)

I find it interesting and concerning that the aptamer for 3-methylxanthine is so short. Maybe the hammerhead ribozyme needs to be added to the sequence. I will check with Dr. Campbell about it.

Also, in my meeting with Dr. Campbell we talked about comparing the structure of theophylline aptamer in Riboswitch D from File:Topp et al. (2010).pdf to the structures of the caffeine aptamer and 3-methylxanthien aptamer in Riboswitch D. M-fold is a web based software that predicts secondary structures of DNA and RNA, which we can use to compare the structures of the aptamers to see if Riboswitch D will work for the other three aptamers.

I read the paper characterizing the software program File:Zuker(2003).pdf to understand what the input and output values mean for the program.

Information on interpreting output results:

Energy dot plot In the upper triangular region, a dot in row i and column j represents a base pair between the ith and jth bases. The dots represent the superposition of all possible foldings within p% of ΔGmfe, the minimum free energy, where p is the maximium percent deviation from ΔGmfe. Different colors are used to indicate varying levels of suboptimality. The number of colors ranges from two to eight (the default). If n colors are used, the first color indicates base pairs in optimal foldings. These base pairs are also plotted in the lower left triangle (reversing row and column) for emphasis. The remaining n-1 colors are used for base pairs in suboptimal foldings. If ΔGi.j is the minimum of the free energies of all possible structures containing base pair i.j, and if ΔGmfe+(k-2)pΔG/(n-1) < ΔGi.j ≤ ΔGmfe+(k-1)pΔG/(n-1), then color k is used for base pair i.j, for 2 ≤ k ≤ n. When n is 8 (the default), the optimal base pairs are colored in red and black colors base pairs that are least likely to form. M-Fold Characterization of the Theophylline, Caffiene, and 3-Methylxanthine Aptamers

M-Fold Characterization of the Theophylline, Caffiene, and 3-Methylxanthine Aptamers

M-Fold Server Input (http://mfold.rna.albany.edu/?q=mfold/RNA-Folding-Form):

I used the RNA Folding Form with no constraints. I kept the default values. I selected immediate job since the sequence is short. Also, I kept the default values for output.

File:Defaultvaluesinput.jg

Default values for user input describing folding conditions.

Default values for user input describing output conditions.

Theophylline Results: Theophylline Sequence:

Sequence output with number of nucleotide bases, max folds, for window size 5. Window size is determined by program based on sequence length.

Energy Diagram. The optimal energy for optimal folding in -71.8kcal/mol. I,j, k, which define the helix are plotted in integer units of kcal/mol.

Curricular structural plots.

Caffeine Results: Caffeine Sequence:

Sequence output with number of nucleotide bases, max folds, for window size 3. Window size is determined by program based on sequence length.

Energy Diagram. The optimal energy for optimal folding in -25.6kcal/mol. I,j, k, which define the helix are plotted in integer units of kcal/mol.

Curricular structural plots.

3-Methlyxanthine Results: 3-Methlyxanthine Sequence:

Sequence output with number of nucleotide bases, max folds, for window size 0. Window size id determined by program based on sequence length.

Energy Diagram. The optimal energy for optimal folding in -8.9kcal/mol. I,j, k, which define the helix are plotted in integer units of kcal/mol.

Curricular structural plots.

I put all the structures of the three aptamers together so we can compare their structures.

Conclusion: None of the aptamers are similar in structure. The second structure of caffeine is slightly similar to the second structure of theophylline. 3-Methylxanthine looks very odd and very different from the rest of the apatmers. I am going to send this information to Dr. Heyer to see if she can help us explain the results.

cadoyle, 26 August 2013

My objective for today is to start designing a method to build riboswitches for caffeine and 3-methlyxanthine.

I can build the riboswitches using Golden Gate Assembly (GGA). Adapting riboswitch D for theophylline from Topp et al. (2010) modified by Becca in the lab during the summer of 2012, I can build riboswitches for the new aptamers.

I looked at Becca's power point and designed a protocol for making a new riboswitch from scratch using GGA.

Outline: 1) Get each part including destination plasmid 2) Perform iPCR on each part 3) Perform GGA 4) Sequence Verify 5) Test

I have a few questions about the method I generated to ask Dr. Campbell: 1) What vector should the riboswitches be cloned into? 2) How do we get the sequence of the aptamer cloned?

Meeting with Dr. Campbell about method to design riboswitches:

The way I had designed the protocol for riboswitch design still utilizes the old method of cloning and PCR. There is no reason to start from scratch to build the riboswtich. We can design primers for anywhere on the existing riboswitch for theophylline and remove the aptamer and replace it with any aptamer we like. We can clone the aptamer upstream of the RBS and screen by size to see if the aptamer is the correct position. Dr. Campbell said that any vector ending in an 8 will work because it has the BSAI removed for GGA.

Therefore, we need to the apatmers sequences for caffeine and 3-methylxanthine to be synthesized. IDT has a product called g-blocks where you can a sequence synthesized but not cloned cheaply. I will pull the aptamer sequences together and send to Dr. Campbell.

What I need to do to move forward: 1) Get sequences to Dr. Campbell for production of G-blocks 2) Design primers to insert aptamers into theophylline riboswitch 3) Design primers to remove theophllyine aptamer 4) I need to send to Dr. Campbell Lee et al. (2010) which I believe contains information on how the location of an RBS effects the affinity of a riboswitch for a metabolite.

I submitted my Thesis Proposal today and should know by Friday if it was approved. Click here to download File:DoyleThesisProposal.doc

cadoyle, 27 August 2013

My objective today is to gather sequences for making G-blocks and design primers to insert the aptamer G-blocks into the existing riboswitch for theophylline.

I emailed Dr. Campbell the Lee et al (2010) paper and he said we needed reference 20 for the theophylline aptamer. Reference 20 is Soukop et al (2000). Relooking at this paper we noticed that there are two aptamers for 3-methylxanthine. From the data it seems that the 3-methylxanthine aptamer with only a C22 has a higher affinity for 3-methylxanthien despite low specificity overall. We decided that it would be better to test both as we are unsure how either will operate in our riboswitch. Therefore, we will have two aptamers for 3-methlyxanthine and 1 for caffeine.

Below is the G-block information and primer design for caffeine, 3-methlyxanthine, and the theophylline riboswitch:

Riboswitch Designs:

Caffeine:

Aptamer Sequence (99mer): ‘5-GGAUGUCCAGUCGCUUGCAAUGCCCUUUUAGACCCUGAUGAGGAUCAUCGGACUUUGUCCUGUGGAGUAAGAUCG CGAAACGGUGAAAGCCGUAGGUCU-3'

Primers to add BSAI:

Caffeine Aptamer For (27mer): GGTCTC A GGAUGUCCAGUCGCUUGCAA BSAI 1bp 20mer of caffeine aptamer

Caffeine Aptamer Rev (27mer): GGTCTC A UGUCCTUCGGCTTTCUCCGT BSAI 1bp 20mer of caffeine apatmer

3-methlyxanthine:

Aptamer sequence (25mer): 5- AUACCAGCCGAAAGGCCAUUGGCAG-3

Primers to add BSAI:

3-MethylC22 Aptamer For (20mer): GGTCTC A AUACCAGCCGAAA BSAI 1bp 13mer of 3-methylxanthine

3-MethylC22 Aptamer Rev (19mer): GGTCTC A CTGCCUUTGGCC BSAI 1bp 12mer of 3-methylxanthine

3-Methylxanthine Protoypic Aptamer Sequence: 5’-AUACCAAGC-GAAAGGCCAUUGGAAG-3’

3-Methylprototypic Aptamer For (20mer): GGTCTC A AUACCAAGC-GAA BSAI 1bp 13mer of 3-methylxanthine

3-Methylprototypic Aptamer Rev (19mer): GGTCTC A CTTCCUUTGGCCT BSAI 1bp 12mer of 3-methylxanthine

Theophylline: ‘5-ggtgataccagcatcgtcttgatgcccttggcagcaccctgct-3’

Theophylline Aptamer For (27mer): GGTCTC A ggtgataccagcatcgtctt BSAI 1bp 20mer of caffeine aptamer

Theophylline Aptamer Rev (27mer): GGTCTC A agcagggtgctgccaagggc BSAI 1bp 20mer of caffeine apatmer

Riboswitch Addition:

BBA_J100065 Gaattcgcggccgcttctagagaaatcataaaaaatttatttgctttgtgagcggataacaattataatagattcaattgtgagcggataacaattactagagatacgactcactataggtaccggtgataccagcatcgtcttgatgcccttggcagcaccctgctaaggtaacaacaagatgctgagacctactagtagcggccgctgcag

Primers to Perform GGA to remove existing aptamer and insert desired aptamer:

For aptamer remover (27mer): GGTCTC A aaggtaacaacaagatgctg BSAI 1bp 20mer of RBS + spacer + GGA prefix

Rev aptamer remover (27mer): GGTCTC A ggtacctatagtgagtcgta BSAI 1bp 20mer of KpnI + Lac Operon

Meeting with Dr. Campbell about Primer design:

Dr. Campbell and I decided that it was better to wait on ordering primers and G-blocks and plan out the design of the riboswitch more carefully. It seems that it is not possible to plug and chug different aptamers into an existing riboswtich but that each riboswitch must be designed methodically.

Our objective is to look at the original paper that characterized the theophylline aptamer and Topp et al (2010) that designed a riboswitch for the aptamer to see if we can rationally design a method to develop a riboswitch for any aptamer.

We generated a few questions about how Zimmerman (1997) developed the original aptamer for theophylline and how Topp et al. (2010) designed a riboswitch for the aptamer: 1) What did Zimmerman do to characterize the apatamer? 2) How did Topp connect the aptamer with the riboswitch? 3) How did Topp get the RBS to fold up and hide in the aptamer? 4) Was the RBS random or specific for the aptamer?

We decided that we could use M-fold to look at the structures of the riboswitches and see why certain designs failed to detect theophylline in Topp et al (2010). Therefore, we are going to try to meet with Dr. Heyer, who is an expert in M-fold to devise a plan. I am going to write Dr. Heyer and email and explain out objectives and send her Topp et al and Zimmerman papers. Also, I will look to answer the following questions 1) Did Topp et al (2010) change the sequence of the previously characterized theophylline aptamer and 2) how did they determine which RBS to use for the theophylline riboswitch.

cadoyle, 28 August 2013

After meeting with Dr. Campbell yesterday 27 August 2013 we decided to write Dr. Heyer and email about getting together for a riboswitch design meeting. I was assigned to write Dr. Heyer and email explaining our goals and questions about rationally designing riboswitches for known aptamers. Below is the email I sent Dr. Heyer with attached PDFs. I am currently waiting Dr. Heyer's response.

Dr. Heyer,

Dr. Campbell and I would like to meet with you to talk about how we can look at the structures of riboswitches for theophylline and determine how they converted aptamers into riboswitches, in hopes to rationally design riboswitches for caffiene, 3-methylxanthine, and xanthine. By looking at the paper Topp et al (2010) we would like to compare and contrast the different riboswitches built and determine 1) how they were able to get the RBS to base pair with the theophylline aptamer and 2) why certain riboswitch structures did not work. We were thinking that we could utilize M-fold to help us understand the differences in the riboswitch designs and how they relate to the folding of the aptamer. In our meeting I will present information on whether 1) Topp et al (2010) changed the sequence of the previously characterized theophylline aptamer and 2) how they determined which RBS to use for the theophylline riboswitch. Attached are three PDF files 1) the Topp et al. (2010) 2) Supplemental for Topp et al (2010) with figures of the designed riboswitches (Becca Evans developed riboswitch D) and methods, and 3) Zimmerman et al. (1997), which originally characterized the theophylline aptamer. Please let us know some times when you are available so we can pick one that works for both of us.

Thanks,

Catherine

Lab Meeting Presentation on Aptamers and Riboswitches:

For Friday's lab meeting (08/30/2013) I am presenting the aims of my project. My main objective is to explain what an aptamer and riboswitch are and how we can use them to detect an unknown metabolite of caffeine.

Aptamer: An aptamer is short nucleic acid sequence that binds to a specific small molecule or ligand.

I found this great video that explained an aptamer as a dart aiming for a specific point on a target. "A Customized DNA dart" [[3]]

Riboswitch: A riboswitch is a regulatory segment of a messenger RNA molecule that binds to a small molecule, resulting in a change in production of the proteins encoded by the mRNA. Characteristics: -Translational control -Contains aptamer sequence -In 5’ untranslated region of mRNA

In my presentation I made two other slides: 1) showing the folding of the riboswitch with the aptamer from Topp et al (2010). 2) a graph showing how different riboswitches detect theophylline in E. coli.

Tomorrow I will finish up power point and post in the lab notebook. I need to add what the goal of my project is and a few more diagrams to explain how riboswitches and aptamers interact.

cadoyle, 29 August 2013

My objective today is to finish lab the presentation and practice the presentation.

I added a slide showing theophylline biosynthesis, caffeine metabolism, and caffeine derivatives with known aptamers.

I will present this final version tomorrow in lab meeting File:LabMeeting08-30-13.pptx

cadoyle, 1 September 2013

My objective today is to read Topp et al. 2010 and Zimmeran 1998 paper to see if I can determine where Topp et al got the RBS used in their riboswitches and if they mutated the aptamer sequence for theophylline.

After Reading Topp et al (2010) and Zimmerman (1998) I found out a few things: 1. The riboswitches tested were generated using a combination of rational design and in vivo screening. 2. Before they started there had already been published theophylline synthetic riboswitches by Lynch et al. (2007). 3. They made a library of random parts and through high-put screening found the riboswitch that optimized the detection of E.coli. 4. The aptamer was not from Zimmerman 1998 but from Zimmeran 2000. It was not mutated. 5. I am still not sure how they choose the RBS but they mention it is important for it to base pair with aptamer 5. Randomized sequences truly are random. 6. Shorter spacing between the RBS and start codon increased specificity.

I am going read two papers by Lynch describing screening protocol and generation of random sequences. Also, I am going to read the paper by Topp and Galivan (2008) that describes a screening method for the riboswithces once you have candidates.

Notes from Lynch et al. (2007): - Lynch uses cassette-based PCR mutagenesis to create 5 different libraries where the distance between the aptamer and the RBS were varied between 4 to 8 bases and the sequence was radonmized fully. It had been shown earlier that longer or shorter spacing resulted in poorly functioning riboswitches.

-Once they had the libraries they tested them using a Miller assay. To be considered a good riboswitch they had to meet four requirments:

(1) showed an activation ratio of greater than 2.0 in two separate determinations (2) displayed a mini- mum level of b-galactosidase activity in the presence of theophylline (an OD420 R 0.04 in the Miller assay, regard- less of cell density) (3) grew normally relative to others in the plate (as represented by OD600) (4) showed consistent results between the two plates. This simple analysis significantly reduced the number of potential candidates, of which greater than 90% were confirmed as functional synthetic riboswitches when assayed individually in larger volumes of culture

- the pairing region between the aptamer and the ribosome binding site is important for riboswitch function.

I need to the read paper on how lynch made the riboswitch (Desaia and Gallivan, 2004).

cadoyle, 2 September 2013

My objective today is to read the original paper that characterized the theophylline riboswitch and make a presentation on how the riboswitch evolved to give to Dr. Campbell and Dr. Heyer tomorrow in our meeting.

Notes: Desaia and Gallivan, 2004: 1. This the first paper published on the theophylline riboswitch 2. Their original design started with trying to insert the mTCT8-4 theophylline aptamer from Zimmerman (2000) 5 bp upstream of the RBS in the 5' untranslated region. 3. The reporter gene used was B-gal 4. The RBS used was Shine-Dalgarno sequence: AGGAGGU 5. To insert the aptamer into the 5' untranslated region they used a primer containing a Kpn1 site, the aptamer, 5bp randomized sequence, and 35 bp overhand of the RBS. 6. To test their riboswitch they transformed the riboswitch into E.coli than plated the cells on media containing theophylline, caffeine, or no small molecule. The riboswitch was more responsive to theophylline but did respond to caffeine. 7. The paper does not tell how but they realized that having the ribsowtch 8bp upstream of the RBS increased the riboswtich specificity.

Notes Lynch et al., 2007: 1. After Desaia and Gallivan published their paper Lynch et al looked to improve the specificity of the ribsowtch and see if they could understand why the riboswitch published by Desaia and Gallivan worked. 2. In the riboswitch by Desaia and Gallivan there is a section of randomized bases. Lynch wanted to see how important these bases are. 3. They used cassette-based PCR mutagenesis to create 5 different libraries in which the distance between the RBS and aptamer varied between 4 and 8 bases and the sequence was completely randomized. 4. Then they performed a high-through put assay where they plated colonies that grew white on X-gal media with no theophylline in cultures o/n containing theophylline. 5. They found two clones that had high B-gal expression in a culture supplemented with theophylline. 6. Both these clones contained the aptamer 8bp upstream of the RBS like Desaia and Gallivan had seen. 7. They also realized that when they used M-fold (They have methods on how they used it) that the RBS base paired with the aptamer and the randomized sequence making the switch in a "off" state. 8. This showed that the RBS being able to bind to the aptamer and their being 8 bp between the RBS and aptamer essential to a functional riboswitch.

Notes: Topp et al. 2010: 1. Now rereading Topp et al. I understand how they came to the design of the final theophylline riboswitch 2. The paper wanted to see if mutating the RBS to make it base pair with the aptamer would increase its affinity. 3. They found that two switches worked best and those switches had more bp to the RBS. They differend in the number of base pairing between RBS and Ribosome

Questions: 1) can you mutate a RBS and it still function? 2) should we use the same RBS? 3) They don't mention why they choose that RBS?

Link to my ppt presentation with figures and data from the papers. File:Riboswitch History.pptx

All the papers I read and used for the Riboswitch History ppt are: File:Desai dissertation.pdf File:Desaia and Galivan Supp.pdf File:DesaiandGallivan(2004).pdf File:Lynchetal.pdf File:Supp Topp(2010).doc File:Topp dissertation.pdf File:Topp(2010.pdf) File:ToppandGallivan.pdf File:Zimmerman1997.pdf File:Zimmerman2000.pdf

cadoyle, 3 September 2013

Today I met with Dr. Campbell and Dr. Heyer and presented my ppt on the Riboswitch History.

Notes from meeting: 1. Dr. Campbell had an idea that we could feed the cells xanthine or 3-methlyxanthine and overload the cells so they will stop theoretically producing xanthine or 3-methylxanthine and go ahead and produce theophylline. It would stop the de-methylation process. 2. After going over the ppt it seems that we can predict the free energy of each aptamer using m-fold. Lynch et al did this to show the on and off switch of the riboswitch for theophylline. 3. There has to be 8 bases between the RBS and aptamer. 4. D riboswitch had 1/2 the base pairing with ribosome than riboswitch E. The specificity of E is greater by it off is not very off. Maybe if we have 5,6, or 7 base pairing the riboswitch would work better.

Plan of action: 1. Verify the results of Lynch et al. using M-fold to see the free energy calculations are correct 2. Design a program that can randomize the 8 bp spacer between the RBS and aptamer creating 65,00 combinations needed for each aptamer. We can run the sequences through M-fold and using the free energy of each known aptamer predict which riboswitch will work. Then we can it synthesized and start testing. 3. In the mean time I will feed the cells xanthine and 3-methylxanthine and see if we get theophylline to be produced. If we give the cells what they might be producing maybe they will make theophylline and not convert it into another metabolite. 4. Also, as a back up there is a riboswitch that can detect 3-methlyxanthien and theophylline and we can use it see if 3-methylxanthine is present.

Tomorrow I will verify Lynch et al. results on M-fold and start writing the pseudo code.

cadoyle, 4 September 2013

My objective today is to run the riboswitches designed by Lynch et al through M-fold to verify the change in free energy they viewed with the riboswitches 8.1 and 8.2 depending on the random bases in the 8bp spacer.

Predicted folding structure of riboswitches 8.1 and 8.2

Computational RNA Folding Protocol Secondary structures of riboswitches were determined using the RNA mFold web server (http://www.bioinfo.rpi.edu/applications/mfold/rna/form1.cgi). Sequences stretching from 5’- end of the theophylline aptamer to 3’-end of the AUG start codon of riboswitches 8.1 and 8.2 were entered and secondary structures were calculated without constraints at 37C with 50% suboptimality.

I confirmed Lynch et al. (2007) results. M-fold yielded 9 and 8 structures respectively. Extending the sequences of 8.1 and 8.2 to the transcription start site increased the number of suboptimal folds, but did not change the structure of the lowest-energy fold. See below for results and figures.

Results for 8.1 Riboswitch:

Inputted sequence:

Results for 8.2 Riboswitch:

Inputted sequence:

File:8.2 seq.jpg

Now that we know that the Lynch et al (2007) protocol works I can start writing the code to randomize the sequences and determine the free energy of different riboswitch designs. The free energy needs to be less than the energy of aptamer ensuring binding of the aptamer to the small molecule.

cadoyle, 4 September 2013

My objective today to is write pseudo code for the program that will determine the free energy of the different riboswitches by randomizing the 8 bp spacer. This will allow us to test 65,000 combinations and pick the optimal ones for testing in the lab.

See below for diagram of how the code will operates

cadoyle, 6 September 2013

My objective today is to talk to Dr. Heyer about issues with implementing the code and downloading M-fold. I will be giving a lab presentation on Riboswitch History in lab meeting today.

Notes from meeting with Dr. Heyer: 1. M-fold is not working because I do have have C++ complier. I will have to use a school computer to download one. 2. I probably will not be able to get every possible combination of a 8bp sequence because it would take to much memory. I am going to use the permutations function to generate combinations. 3. Dr. Heyer did not think objects were a good idea.

Dowloading C++ complier: I tired to download the complier but it would not install. I going to have to use a school computer.

cadoyle, 8 September 2013

My objective today is to write all the code expect the part that implements M-fold. Tomorrow I will use M-fold on a school computer.

I got the sequence for the 16sRNA to use in my code from [[4]].

I was able to get a randomize function to make a random number of combinations for the riboswitch sequence. My computer is not bale to perform every combination because it does not have enough memory.

I went the lab to see if the computers there had X-code so I can install M-fold. X-code needs to be updated the computers and I could not install M-fold. I emailed Dr. heyer about getting access.

The code is working except for the part that calls M-fold. I will go tomorrow to the computer lab to try to get M-fold to run.

cadoyle, 9 September 2013

I got access to the computer lab in chambers and will go today to download M-fold.

I downloaded M-fold and successfully installed the program. I tried running the program from the python script and from command line. I was not able to get the program to run. I re-read the manual and googled how to run the program, but could not figure it out. I emailed Dr. Heyer to see if she can help me. I am waiting her response. I will try again tomorrow.

cadoyle, 10 September 2013

Dr. Heyer emailed me about placing ./ in front of the script when I am trying to run on command line. M-fold still did not run when i tried using ./so I emailed her again. I waiting to see if she can meet with me.

I had a meeting with Dr. Campbell today here are the notes: -Xanthine and 3 methylxanthine arrived. -Xanthine is stored at room temperature and 3-methylxanthine is stored at 4C - I will feed the cells 0.5mmM of 3-methlyxanthine and xanthine with 0.5mmM of caffeine and see if we can get the cells to produce theophylline. - Also, we talked about generating a semester long selection process: 1. Putting the riboswitch with a fitness module we could see if e.coli can mutate over time to produce theophylline. 2. I will read about E.coli mutation rates in liquid vs. plate and see if I can design an experiment to test the evolutionary process of e.coli. We will not know how long it will take for E.coli to make theophylline but is worth a shot. If E. coli does produce theophylline we can sequence the genome and then build riboswitches for that sequence.

Tomorrow I will grow liquid cultures of e.coli with the riboswitch and ecdm8 with caffeine, xanthine and 3-methlyxanthine. See figure below. We have three different options for how the riboswitch and ecDM8 can intereact. 1. the riboswitch is on a high copy plasmid, so it will produce more GFP 2. the ecDM8 gene is on the high copy plasmid and should produce more enzyme for converting caffeine to theophylline, 3. both the riboswitch and ecDM8 on the same high copy plasmid. I will test each type of cell in the different media.

cadoyle, 11 September 2013

Dr. Heyer and I meet today. M-fold needs Fortran complier which the school computers do not have. On Friday she will help me install the Fortran complier. Dr. Heyer, also told me that I wrote my program wrong and it will not give every combinations. I will try to rewrite it by Friday.

I started to plan out how I would make all the media to grow E.coli with the riboswitch + ecDM8 to see if we can force the cells to make theophylline by flooding the cell with 3-methlyxanthine or xanthine, which are downstream derivatives of caffeine.

Media: 50mM Xanthine:

We found that xanthine is slightly soluble in water but is freely soluble in NaOH.

I will make 2mls of 50mM Xanthine in 10mM of NaOH

5mM *(1/1000) = 0.05M 2mLs (1/1000) = 0.02L 0.05M * 0.02L = 1 X 10^-4mol 1X 10^-4mol * 152.11g/mol = 0.015g of xanthine in 2ml of NaOH

50mM 3-Methlyxanthine:

We found that 3-methlyxanthine is slightly soluble in water but is freely soluble in NaOH.

I will make 2mls of 50mM 3-methlyxanthine in 10mM of NaOH

5mM *(1/1000) = 0.05M 2mLs (1/1000) = 0.02L 0.05M * 0.02L = 1 X 10^-4mol 1X 10^-4mol * 166.14g/mol = 0.017g of 3-methlyxanthine in 2ml of NaOH

50mM Caffeine:

I will make 20mls of 50mM Caffeine

5mM *(1/1000) = 0.05M 20mLs (1/1000) = 0.02L 0.05M * 0.02L = 1 X 10^-4mol 1X 10^-4mol * 194.2g/mol = 0.194g of caffeine in 20mls of H20

10mM NaOH:

I will make 10mls of 10mM NaOH from 5M stock

10mM *(1/1000) = 0.01M 10mLs (1/1000) = 0.01L (0.01M)(0.5L) = (5M)(x) x= 100ul of 5M NaOH in 49.9mls of dH20

Tomorrow I will make these solutions and inoculate the media with the cells containing the riboswitch and ecdM8 gene.

cadoyle, 12 September 2013

My objective today is to make solutions for inoculations and finish the program.

I made media according to calculations on 11 September 2013. I filter sterilized each solution.

I added antibiotics to LB to make media acceptable for the different high and low copy antibiotic plasmids:

LB + Amp (high copy): Amp (high copy): 100ul in 200mls

LB + Amp (high copy) + chlor (low copy): Amp (high copy): 100ul in 200mls Chlor (low): 147ul in 200mls

LB + Amp (low copy) + Chlor (high copy) Chlor (high): 1000ul in 200mls Amp (low): 40ul in 200mls

Flooding Cell Experiment:

For each cell type there are 6 different experimental conditions: 1) 20ul caffeine + 20ul of dH20 + 2ml LB + antibiotic 2) 40ul of dH20 + 2ml LB + antibiotic 3) 20ul caffeine + 20ul xanthine + 2ml LB + antibiotic 4) 20ul xanthine + 20ul of dH20 + 2ml LB + antibiotic 5) 20ul caffeine + 20ul 3-methlyxanthine + 2ml LB + antibiotic 6) 20ul 3-methlyxanthine + 20ul of dH20 + 2ml LB + antibiotic

I inoculated the following cells into the proper media:

1) J10015 on psB1A8 (high copy): contains ecDM8 + Riboswitch D - Location: Box 5-2 #3 - test tubes are labeled 1-6

2) J119303 on psB4C5 (low copy) + J100079 on psB1A8 (high copy): contains ecDM8 (J119303) +Riboswitch (J100079) - Location: Box 2-4 #83 - test tubes are labeled 7-12

3) J119304 on psB1A8 (high copy) + J100079 on psB4C5 (low copy): contains ecDM8 (J119304) +Riboswitch (J100079) - Location: Box 4-4 #83 - test tubes are labeled 13-18

I grew the cells over night at 37C.

Tomorrow I measure GFP expression of each cell type in the different media conditions

cadoyle, 13 September 2013

My objective today is to tweak the program and measure the GFP fluorescence in each culture I grew overnight to see if theophylline is being made by E.coli.

The program is now working and will produce 65,536 riboswitch combinations. I need to talk to Dr. Heyer about buying Una-fold. After we get Una-fold downloaded I can calculate the free energy and get putative riboswithces.

I measured the GFP fluorescence for each culture grown o/n at 37C that was inoculated on 09/12/13 following this protocol [[5]]

Results:

Here is my lab presentation with a summary of my work this week in the lab:

File:LabMeeting09-13-13.pptx

cadoyle, 14 September 2013

Flooding Cell Experiment Replicate #2: For each cell type there are 6 different experimental conditions: 1) 20ul caffeine + 20ul of dH20 + 2ml LB + antibiotic 2) 40ul of dH20 + 2ml LB + antibiotic 3) 20ul caffeine + 20ul xanthine + 2ml LB + antibiotic 4) 20ul xanthine + 20ul of dH20 + 2ml LB + antibiotic 5) 20ul caffeine + 20ul 3-methlyxanthine + 2ml LB + antibiotic 6) 20ul 3-methlyxanthine + 20ul of dH20 + 2ml LB + antibiotic 7) 20ul 3-methlyxanthine + 20ul of dH20 + 20ul of xanthine + 2ml LB + antibiotic 8) 20ul 3-methlyxanthine + 20ul of dH20 + 20ul of xanthine + 20ul of caffeine + 2ml LB + antibiotic

I inoculated the following cells into the proper media: 1) J10015 on psB1A8 (high copy): contains ecDM8 + Riboswitch D - Location: Box 5-2 #3 - test tubes are labeled 1-6 2) J119303 on psB4C5 (low copy) + J100079 on psB1A8 (high copy): contains ecDM8 (J119303) +Riboswitch (J100079) - Location: Box 2-4 #83 - test tubes are labeled 7-12 3) J119304 on psB1A8 (high copy) + J100079 on psB4C5 (low copy): contains ecDM8 (J119304) +Riboswitch (J100079) - Location: Box 4-4 #83 - test tubes are labeled 13-18 I grew the cells over night at 37C.

Tomorrow I measure GFP expression of each cell type in the different media conditions

cadoyle, 15 September 2013

I read the GFP expression of cultures made on 15 September 2013. See complied results on 16th September 2013.

Flooding Cell Experiment Replicate #3: For each cell type there are 6 different experimental conditions: 1) 20ul caffeine + 20ul of dH20 + 2ml LB + antibiotic 2) 40ul of dH20 + 2ml LB + antibiotic 3) 20ul caffeine + 20ul xanthine + 2ml LB + antibiotic 4) 20ul xanthine + 20ul of dH20 + 2ml LB + antibiotic 5) 20ul caffeine + 20ul 3-methlyxanthine + 2ml LB + antibiotic 6) 20ul 3-methlyxanthine + 20ul of dH20 + 2ml LB + antibiotic 7) 20ul 3-methlyxanthine + 20ul of dH20 + 20ul of xanthine + 2ml LB + antibiotic 8) 20ul 3-methlyxanthine + 20ul of dH20 + 20ul of xanthine + 20ul of caffeine + 2ml LB + antibiotic 9) 20ul 3-methlyxanthine + 20ul of dH20 + 20ul of xanthine + 2ml LB + antibiotic 10) 20ul 3-methlyxanthine + 20ul of dH20 + 20ul of xanthine + 20ul of caffeine + 2ml LB + antibiotic

I inoculated the following cells into the proper media: 1) J10015 on psB1A8 (high copy): contains ecDM8 + Riboswitch D - Location: Box 5-2 #3 - test tubes are labeled 1-6 2) J119303 on psB4C5 (low copy) + J100079 on psB1A8 (high copy): contains ecDM8 (J119303) +Riboswitch (J100079) - Location: Box 2-4 #83 - test tubes are labeled 7-12 3) J119304 on psB1A8 (high copy) + J100079 on psB4C5 (low copy): contains ecDM8 (J119304) +Riboswitch (J100079) - Location: Box 4-4 #83 - test tubes are labeled 13-18 I grew the cells over night at 37C.

Tomorrow I measure GFP expression of each cell type in the different media conditions

cadoyle, 16 September 2013

I measured the GFP expression of cultures inoculated on the 15th of September 2013. I combined all three replicates into a excel file and graphed the results. See Excel document below. File:GFPFlourescence09-13-16.pptx

I read some articles about E.coli mutation rates and found that 6.64 generations a day for E.coli. It takes 30 generations for a mutation to occur. There are 0.0024 genomic mutations per generation. It would take 40,000 generations for 100 base substitutions. However, these mutation rates are not under selective pressure with built in fitness. One lab at MSU has been testing e.coli mutation rates and has a protocol for mutating e.coli. Their protocol [[6]] takes place over 75 days for accumulation of 500 generations. I was thinking that we could run the experiment for 35 days instead. Also, I think we could do 3 tubes of 5mls for ECDM8 + Riboswitch instead of 1 10ml flask. Each day I could transfer 100ul form each tube into a fresh tube, measure OD and GFP fluorescence, and plate 100ul of plates to detect contamination.

cadoyle, 17 September 2013

Today I meet with Dr. Campbell. Notes for Meeting:

1. Dr. Campbell requested the order for Unafold, which we can use to generate the free energy of each riboswtich sequence. Hopefully it will be here by Friday so we can get started on testing the sequence combinations.

2. We are going to try to perform the Evolution experiment, except we are going to try one with the TetA resistance fitness module and one with the ADHE fitness module. I will outline both protocols and post tomorrow or Thursday in a pptx for lab meeting. - Note: Riboswitch + TetA cells are in box #3-4 #88,89 we have them in psB4C5 and psb2K3

3. Dr. Campbell and I talked about how we would need to determine genomic mutations vs plasmid mutations as well. I will outline in lab presentation.

4. From the Flooding experiment on 09/16/13 it seems that eCDM8 might not be translated or transcribed. We could do a western blot to see if the gene is being translated in E.coli. I will need to look up antibodies and eptitote tags that we can use.

cadoyle, 18 September 2013

My objective today is to draw out the methods for the evolution experiment and make a power point of the procedure for lab meeting. I made the power point see below and sent it to Dr. Campbell. We had multiple email converstaions and decided to email the ppt to. Dr. Eckdhal with the questions we have concerning the experimental controls and media used.

Dr. Eckdahl,

Dr. Campbell and I are planning an experiment where we see if E.coli can naturally mutate over time into mutants that produces theophylline with the tetA and AdhE fitness modules. I have attached a ppt of the experiment. Dr. Campbell and I would like your input on some of the questions we have about controls and the experiment.

Notes About ppt:

I made a powerpoint about the evolution experiment. The first slide is results from the caffeine derivative experiment. The next six slides show plasmid constructs to be transformed into JM1009 and AdhE- cells respectively. Then I show how I will grow 3 tubes of 5mLs with each type of cell and a control without tetA. I make a note about having to test the amount of TetA to add over time. I thought 0.005mMl every three 3 days. The max I believe is 0.05mM that E.coli can handle so we would reach the max at day 36. I will have to play around with it though. Then to see if the mutation happened in the plasmid or genomic DNA I can grow the cells glowing green on chlor+ Amp with not TetA and then grow those colonies that grow on chlor+Amp+tetA.

We have questions about:

Whether we need a control where cells lacking tetA gene are treated the same way?

Whether we need a control where adhE-cells are lacking the adhE plasmid?

Is liquid better than plates?

Should they be grown at 37C or room temperature?

Thanks, Catherine

I will present this pptx in lab meeting and get the labs opinion. File:Lab Meeting 09-21-13.pptx

Also, Dr. Campbell sent the check for Unafold today so we are waiting for the program to arrive so we can start testing switches.

cadoyle, 19 September 2013

My objective today is the look up eptitote tags for detecting if ecdM8 is being translated.

I found these three putative tags. His requires a western blot and GFP and CAT do not. GFP can be detected through fluorescence and CAT can be detected through another assay. I think GFP tag would be the best but sent the information to Dr. Campbell and will see what he thinks.

Price for Monoclonal $294.00 from sigma aldrich Gel is $125.00 from Thermo Scientific

Price for Monoclonal $285.00 from sigma aldrich

Price $317.00 from signma aldrich

Dr. Campbell emailed me back about using e-myc tag. I think we will use e-myc because Dr. Campbell has worked with it and know that it works.

We can order the tag from http://dshb.biology.uiowa.edu/c-myc.

Here is the full amino amino acid sequence for myc:

>sp|P01106|MYC_HUMAN Myc proto-oncogene protein OS=Homo sapiens GN=MYC PE=1 SV=1 MPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIWKKFELLPTPP LSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVTELLGGDMVNQSFICDPD DETFIKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKDSGSPNPARGHSVCSTSSLYLQD LSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTESSPQGSPEPLVL HEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAGGHSKPPHSPLVLKRC HVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQISNNRKCTSPRSSDTEENVKRRTHN VLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAEEQKLISEEDLL RKRREQLKHKLEQLRNSCA

I wil generate a 10bo eptitope tage fromt the sequence based of one Dr. Campbell's papers [[7]]

cadoyle, 20 September 2013

My objective today is to generate the epitope tag sequence for c-myc and develop primers for cloning it downstream of ecdM8.

I based the epitope sequence tag off of Dr. Campbell's paper. I generated a G-block sequence with BsaI sites to by synthesized and primers for GGA and the traditional way of cloning. File:Epitopeprimers.docx

Also, I presented my lab presentation today about the evolution experiment. File:Lab Meeting 09-21-13.pptx

I got great feed back:

1. We all agreed that having all the controls is the most pragmatic and would make the most sense. One can never have to many controls and helps us argue for programmed evolution through the fitness modules.

2. We decided that plates would be a better medium to perform the experiment on. The diffusion of caffeine across the plate could encourage natural selection pressure. Some areas will have higher tetA which could cause a higher number cells to be rewarded in that area. Also, we will be able to visualize what cells produced the mutation unlike in culture where we have a mixture of cells.

3. It would be beneficial to see how low can we go and still detect theophylline with the riboswitch + GFP gene. For example, If the entire 2 mL has to be 50 µM, then the cells may not be able to produce that much theophylline. Perhaps the flow cytometer will detect what we have not so far.

4. I am going to look back at Will DeLoache's thesis where he quantified diffusion on different percent agar plates to see if we can get an insight into how diffusion works on agar plates.

cadoyle, 21 September 2013

My objective today is to read the paper Dr. Campbell sent File:Sb4000146-2.pdf about ecdM8 in E. coli. Also I will look back through the development of the theophylline aptamer to see if the riboswitch ever showed an affinity for 3-methlyxanthine.

Earlier today Dr. Campbell sent an email about diffusion on plates:

I was playing with Micah's model today trying to get a sense of what might be going on with theophylline production.

The question I am grappling with is the diffusion rate of theophylline out of the cells. In Micah's model, the media is not shaken, so cells and theophylline accumulate locally. When I adjusted the settings to have fast diffusion and not much difference in growth or energy levels, the cells almost all die out, but the same event saves the makers every time. A few cells will collect a critical mass of cells and theophylline in a small area, and these survive and thrive. This makes me more convinced that we need to do these experiments on plates instead of liquid media. Can we grow cells on plates in some way to let theophylline accumulate enough to produce detectable GFP? What concentration would we need in plates to detect GFP by eye? If the colonies are really small, we would never see GFP. Is this true for a lawn of bacteria as well?

I know the MWSU students did some plate experiments in the summer of 2013. Todd, could you summarize what your students did this summer and what they found? Did they ever make plates with caffeine added to the molten media, or did they only use disks? Also, did they use all three types of constructs including when eCDM8 was on pSB1An and so was the riboswitch+GFP?

I re-read Will DeLoache's thesis about diffusion rates on agar plates. I had forgotten that the counter-intuitive result was due to the effect agar concentration has on the effectiveness of the antibiotic. High agar concentrations makes ampicillin not work as well. It was not diffusion rate after all.

Dr. C.

We are waiting on the results from MWSU they should arrive Monday.

It does seem clear that we should use plates to perform the directed evolution with E. coli. From Dr. Campbell's email it does not seem that Will DeLoache's thesis will be helpful in determining the diffusion rate. Maybe I could do a similar experiment with caffeine or theophylline for the riboswitch + GFP. I will talk to Dr. Campbell about it.

Notes From Quandt et al. (2013):

-The paper tried to use N7-demethlayion to convert caffeine to xanthine by using a caffeine degradation operon in E.coli.

-They discovered that glutathione S-transferase from Janthinobacterium sp. Marseille was necessary to achieve N7- demethylation activity. E. coli cells with the synthetic operon degrade caffeine to the guanine precursor, xanthine.

- The paper noted that previous attempts to express and characterize NdmC in E. coli were also unsuccessful.

- They reasoned that a missing activity supplied by another protein could be essential for N7- demethylation activity, explaining the lack of full functionality of our synthetic operon in E. coli.

-NdmC had previously been found to copurify with an uncharacterized putative glutathione S-transferase (GST) encoded by orf8 in the CBB5 gene cluster.

-Therefore, thy added gst9 to their operon to see if it restored the activity of of NdmC.

- The addition of gst9 enabled growth for the guaB strain on minimal media agar plates supplemented with caffeine or theobromine.

Dr. Campbell and I agree that we need to clone gst9 into our ecdM8 construct. This could be why ecdM8 might not be translated. A western blot will show whether translation is occurring or not.

I found the gst9 sequence and will have to synthesized. I also developed primers for insertion the gene into the construct.

>ENA|ABR90275|ABR90275.1 Janthinobacterium sp. Marseille glutathione S-transferase ATGATCACGCTTTATGACTATGAACTCTCGGGGAATTGCTACAAGGTGCGGATGCTCCTT TCCATCCTGAAGCTACCGTACACGATAGAGACAGTGGAATTTTTCCCGTCCAGGGAACAT AAATCACCGGCCTTCCTGCGCATCAATCCACTCGGCCAGTTGCCGGTATTGCGCGATGAT GATTTCGTGTTGCGCGATGCCCAGGCCATCCTGGTTTACCTGGCGAACAAATACGACACC AGTTTGACCTGGTATCCGACACATGCGCCCGCCATCCTCGGCCAGGCAAATATGTGGCTC GCGTTTGCCGATGGTTTGACCGGCTCGATTTCCGCTGCACGTTTGCATGATTTGTTTTTT TACGATTTCAATGCAGAGCAATGCCGTGCCCGCGCGCACGAACTACTGCGCATACTGGAC GAACACCTGTGGGCGGCGGAACAAAATGGACATCAATGGCTATGTCCGGCCGCCGCACCC ACCATCGCGGATCTTGCCTGCTTCCCGTATATCGCATTGTCGGATGAAGCCGGCGTTTCA CTACTCGACTACCCGGCGGTACGACGCTGGCTGGACCGTGTCAAACGCATCCCCGGATTC ACCGTCATGTCGGGGATTTTTCCAACCTCAGCTTCCTTCGAGCCGGCCTCACTTGCGGAA GAAACCTGTCAATAA

File:Epitopeprimers.docx - Primer Document

I also, looked back at the paper that originally characterized the theophylline riboswitch before Topp et al. tweaked the ribsowitch to have a greater on and off state and the theophylline riboswtich does bind to 3-methylxanthine. Data came from Desai and Gallivan 2004

Figure 6. Genetic selection experiments for E. coli harboring either the wild-type synthetic riboswitch grown in liquid media with increasing concentrations of chloramphenicol. Data are recorded after 18 h of growth at 37 °C. OD600 is a measure of the cell density of the cultures.

This explains why in the caffeine derivative experiment GFP expression increased when 3-methlyxanthine was added to the cultures (See Lab meeting ppt 09-21-13 or data from 09-17-13).

cadoyle, 22 September 2013

My objective today is to set up an experiment to find out how low can we go and still detect GFP expression in 2mL cultures containing theophylline.

I tested the GFP expression at 0, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mM of Theophylline in a 2mL liquid culture. I made 3 X 2mL cultures for each concentration based off the following calculations:

The table's caption

Concentration mM	25 mM Stock (ul)	Lb + Amp	dH20 (ul)
0	0	2	40
0.05	4	2	36
0.1	8	2	32
0.2	16	2	24
0.3	24	2	16
0.4	32	2	8
0.5	40	2	0

I inoculated each tube with J100079 (Riboswitch + GFP) in psB1A2 and grew O/N at 37C [[8]]

Tomorrow I will measure the GFP expression of each culture.

cadoyle, 23 September 2013

My objective today is to measure the GFP expression of each culture from 09-22-13 and analyze the data.

I measured the GFP expression of cultures inoculated on the 22th of September 2013. I combined all three replicates into a excel file and graphed the results. See Excel document below. File:GFPFlourescence09-13-16.pptx

It seems that the flouremeter can detect theophylline at each concentration. This indicates that the construct is quite robust and that if theophylline is present we should be able to detect it.

Results: File:GFP-09-23-13.xlsx

Also, I resubmitted my thesis proposal with corrections. File:DoyleThesisProposal.doc

MWSU sent the data from theophylline experiments on plates see blow. The performed the experiment with the riboswitch + GFP on a low copy plasmid. I would like to try the same experiment with the GFP reporter on a high copy number plasmid. I will talk to Dr. Campbell about it.

File:July 5. CDM clones with different plates.pptx

File:CDM picture results.pptx

UnaFold arrived today. I will try to install tomorrow

cadoyle, 24 September 2013

My objective to day is to install unafold and plan an experiment to test how much theophylline needs to be present for GFP to be visible on plates.

I downloaded unafold. But have not tested the installation. I will test the installation tomorrow. From the print out it seems that it was installed properly. I will test Riboswitch D from Topp et al. as a control to make sure the program is working.

Today I also meet with Dr. Campbell. Here are my Notes:

We are not going to test the translation of ecdM8 with an eptitote tag. It diverges from the goal. It would be more beneficial to clone gst9 upstream of ecdM8 and see if the cells can process caffeine using GFP expression as a reporter. Ben or a student at MWSU will work on cloning gst9.

We are going to try a similar experiment to MWSU on plates with different theophylline concentrations. However, we will also look at the concentration of tetA and theophylline together to get an idea of how we can better construct our evolution experiment with E.coli.

We are going to ask how much theophylline do cells need to grow with a fixed amount of tetA. I will make a ppt to show the experiment and post today or tomorrow for lab meeting.

cadoyle, 25 September 2013

My objective today is to test UNAFold and to start cultures to test how much theophylline is need to grow with a fixed amount of tetA.

Dr. Campbell and Abbey made plates for me. Therefore, I can start the experiment following the outline in the lab meeting ppt (see 27 September 2013 entry)

I inoculated one Lb + Amp culture with J1190079 and one Lb + Amp + .1mm Theophylline with J119140. I grew the cultures O/N at 37C.

Tomorrow I will use a sterile loop to streak for colony isolation on the plates and see if we can detect GFP expression by eye and cell growth.

UNAFold: UNAFold is installed properly but cannot find ss-mm.pl program. The program is installed but it cannot execute the program. The program is in C++. I will try checking the read out from the installation to see if something was installed wrong. If I cannot figure it out I will send Dr. Heyer and email.

cadoyle, 26 September 2013

My objective today is to figure out what is wrong with una-fold and than streak plates for theophylline experiment.

I tried to re-install una-fold. When I did I got an recursive error. I looked it up online and it means that the fortran complier is not compiling. The computer does not have the fortran complier so I emailed Dr. Heyer about installing the complier. I am waiting her response.

I streaked the cultures with a sterile loop on to plates contain 0, 0.05, 0.1, 0.2, 0.4 mmM of theophylline +Tet and +/- Amp. See ppt on 27 September 2013 for protocol. I grew the cultures o/n at 37C. Plates with tetA were covered in foil to prevent degradation. Tomorrow I will see if GFP expression is visible and if cell growth is visible on plates with tetA.

cadoyle, 27 September 2013

My objective today is to analyze the results from the theophylline experiments.

Looking at cell growth of cells with the promoter+riboswitch+GFP on plates with various concentrations of theophylline It seems that GFP expression is slighlty detectble at 0.05mM and increases as the theophylline concentration increases.

Looking at cell growth of cells with the promoter+riboswitch+tetA on plates with various concentrations of theophylline, a fixed amount of tetA with and without AMP It seems that cell growth is detectable at 0.05mM and increases as the theophylline concentration increases.

This indicates that the lowest concentration of theophylline for good yield of expression is 0.1mM. Also, the production of the reporter gene is robust in the presence of the theophylline. This indicates that ecdM8 probably is not translated or we would see some kind cell growth or GFP expression.

Here is my lab presentation with the protocol and results File:LabMeeting092713.pptx

cadoyle, 28 September 2013

My objective today is to start working on my thesis preview presentation.

I put together a draft and sent it to Dr. Campbell. He sent me back some comments and a more concrete outline of how I should organize the presentation.

1) Define the problem with microbial production of drugs. Case study of theophylline from caffeine (used for asthma treatment) 2) Propose the solution of Programmed evolution but only at the conceptual level (tie selection pressure to production of desired compound). 3) Describe how fitness module should work (keep at conceptual level) 4) describe riboswtich function and contains aptamer (I have an animation in Keynote if you want to use that) 5) show how existing riboswitch works with GFP as reporter (show data) 6) Now bring in eCDM8 mechanism and desired outcome (show structures and loss of methyl groups) 7) Show that we never measured theophylline using riboswtich + GFP 8) Show possible problems of too much demethylation. (show structures of all family members) 9) Can we convert known aptamers into riboswitches? (show which two aptamers we are working with) 10) Your research goals: make two riboswitches from aptamers.

This gives you about 1 minute per slide = ten minutes.

Tomorrow I will make some corrections and talk to Dr. Campbell on Monday about more revisions.

Also, I wrote up two reviews of the speaker presentations for my thesis of visiting professors and submitted to Moodle to complete my thesis package.

cadoyle, 29 September 2013

My objective today is to edit presentation and design primers for cloning tetA fitness module downstream of ecdM8.

I designed primers and sent them to Dr. Campbell. File:EcdM8+TetAPrimers1.docx

Dr. Campbell and I will meet tomorrow to go over them.

cadoyle, 30 September 2013

My objective today is to work more on the presentation and work on trying to figure out how to get una-fold to print to the command line and not to a output file.

I looked online about how to get UNA-fold to print to the command line and found a paper by the authors. I am going to read the paper and play around with commands and the redirection of files to see I can simplify the process so I can use subcommand to call UNA-fold from the python script.

I talked to Dr. Campbell about the primers and we want to switch the direction the primers will operate. We want to clone ecdm8 downstream of tetA instead of the reverse. We used [[9]] to generate the primers.

The output from the program was

I checked the primers generated using this word file File:EcdM8+TetAPrimers.docx and sent them to Dr. Campbell for ordering.

Dr. Campbell gave me some slides and animations to use for the presentation. I will play around in combining what I made and he made to finish the presentation.

cadoyle, 1 October 2013

My objective today is to practice the presentation and read the paper about una-fold.

I meet with Dr. Campbell today we reviewed my presentation. The slides are set and I just need to practice. Dr. Campbell and I talked about the data that arrived from MWSU. MWSU had the construct where ecdm8 and the Riboswtich + tetA are in the same plasmid. However, they never got any growth in the presence of tetA. They cloned the riboswitch + tetA downswtream of ecdm8. The primers I designed clone ecdm8 downstream of tetA. It might make difference whether ecdM8 is upstream or downstream. When the primers arrive I will work on cloning ecdm8 downstream of the riboswitch + tetA. In the mean time I will test the construct MWSU designed J10011 to see if I can get expression. It is possible they used to much tetA in their experiment. From their data it seems that maybe having tetA fitness module on a different plasmid than ecdm8 might provide more tetA expression. I will test both constructs: 1) fitness module and ecdm8 on separate plasmids 2) fitness module and ecdm8 on the same plasmid.

Data from MWSU:

File:Powerpoint.pptx File:PlatePowerpoint.pptx

cadoyle, 2 October 2013

My objective today is start cultures of J10011 and J10140 + J119303 for testing ecdm8 with the tetA fitness module in the presence of theophylline.

I added 8ul of 25mM theophylline to a 2ml culture of Lb + AMP(high) and to a 2ml culture of Lb + AMP(high) + Chlor(low) to make 0.1mM theophylline + LB solution.

I inoculated the LB + Amp culture with J10011 (ecdm8 + tetA fitness module on same plasmid) [[10]] and LB + Amp + chlor with J110140 + J110130 [[11]].

I grew the cultures O/N at 37C. Tomorrow I will use a sterile loop to streak the cultures on tetA plates with various concentration of theophylline.

I read the paper on getting UNA-Fold to print to a different file or the command line. It was not very helpful. It wanted you to download a perl script. I believe UNA-fold already has it but will check tomorrow. I am not sure how to get UNA-Fold to print to the command line. I will play around with it tomorrow.

Also, I started working on my lab presentation for Friday. I took the data from MWSU we received on the 10-1-13 and made graphs so the data was easier to read. I also changed the figure legends from the part number to what was actually in the parts so it is easier to conceptualize.

cadoyle, 3 October 2013

My objective today is to streak out cultures on plates with theophylline and tetA to see if with fitness module there is expression.

Using a sterile loop I streaked cultures with J10011 and J110140+J119303 for colony isolation on plates with 10ug/ml of tetA and theophylline concentration ranging from 0-0.4mM.

I placed the plates in the incubator wrapped in foil O/N at 37C.

cadoyle, 4 October 2013

My objective today is to view the results of the colony isolation from yesterday and to take pictures. Also, will finish lab presentation.

Results: J10111 showed no growth at any concentration of Theophylline.

J190403 + J190303 (high promoter ecdm8 (low copy) + tetA fitness module ( high copy) showed growth on plates with 0.05-0.4mM of theophylline.

I think we should test 3-methlyxanthine and xanthine with the J190403 + J190303 construct and see if there is growth.

See lab presentation for pictures of growth.

File:LabMeeting10-4-13.pptx

cadoyle, 6 October 2013

I found a way to copy all the output from M-fold to a new file so I can sort through the file and look for delta G's lower than the energy needed for the apatmer to bind to a ligand. I can use doyle:Desktop catherinedoyle$ cat test2.txt >> test1.txt

I can use this command to copy all files to with dg.txt to a new file: cp *.txt newdir Then I can copy the file contents of each file into a new file. I can than sort the file for delta G's lower than the energy needed for the apatmer to bind to a ligand

I still need to figure out how to call it from the python script and how get spaces between the entries so you know what sequence gave what delta G.

cadoyle, 7 October 2013

My objective today is to practice my presentation for tomorrow an start cultures for testing the tetA fitness module with caffeine, 3-methylxanthine, and xanthine.

I inoculated a 2ml Lb AMP + Chlor culture with J110140+J119303 with 8ul from box 2-4 #93. I grew the culture O/N at 37C shaking.

cadoyle, 8 October 2013

Today I am giving my thesis preview presentation. File:DoylePreview1.ppt

Also, I used a sterile loop and plated the culture of J110140+J119303 on Lb tet, LB tet + Caffeine, LB tet + 3-methlyxanthine, LB tet + xanthine. I covered the plates in foil and placed in the 37C incubator O/N. Tomorrow I will look for growth to see if the riboswitch can detect 3-methlyxanthine and xanthine.

Dr. Campbell and I meet and we discussed that we need to place ecdm8 downstream of teta fitness module so when gst9 arrives we can clone it into the construct and be ready to test the construct in programmed evolution. I will start on this week and finish after fall break. I will also try to work on the program and have questions for Dr. Heyer on Friday.

cadoyle, 9 October 2013

My objective today is to check the results of the experiment yesterday and see if there is cell growth.

I check the plates. None of the plates showed cell growth. I forgot to do a positive control with theophylline + tetA so will redo the experiment with the positive control.

I inoculated a 2ml Lb AMP + Chlor culture with J110140+J119303 with 8ul from box 2-4 #93. I grew the culture O/N at 37C shaking.

Also, I inoculated a 2ml LB + Amp culture with j119140 and one 2mL LB = AMp culture with J119303. I grew the culture O/N at 37C shaking. Tomorrow I will miniprep and the DNA so I can start PCR.

cadoyle, 10 October 2013

I minipreped the cultures of J119140 and J119303 following this protocol [File:http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_MiniPrep.html].

I nanodroped the DNA and got the following results: J119140: 717.92ng/ul A260=14.25 A280= 7.569 260/280 = 1.90 260/230=2.19 J119303: 211.90ng/ul A260=4.238 A280= 2.226 260/280 = 1.90 260/230=2.21

Next, I will perform PCR with J119140 and J119303 to prepare for GGA.

PCR in preparation for GGA: J110140 Master mix 50ul FWD Primer 1ul REV Primer 1ul DNA 1ul Water 47ul

J119303 Master mix 50ul FWD Primer 1ul REV Primer 1ul DNA 1ul Water 47ul

PCR Cycle for J110140 Step 1: 95° C 5 minutes (denature template) Step 2: 95° C 30 seconds (denature dsDNA) Step 3: 61° C 30 seconds (Tm minus 5 degrees) Step 4: 72° C 3 minutes Step 5: Repeat Steps 2 through 4, 29 more times Step 6: Store at RT°

PCR Cycle for J119303 Step 1: 95° C 5 minutes (denature template) Step 2: 95° C 30 seconds (denature dsDNA) Step 3: 57° C 30 seconds (Tm minus 5 degrees) Step 4: 72° C 1 minutes Step 5: Repeat Steps 2 through 4, 29 more times Step 6: Store at RT°

After pCR I removed the tubes and stored in the fridge for GGA after fall break

Also, I used a sterile loop and plated the culture of J110140+J119303 on Lb tet, LB tet + Caffeine, LB tet + 3-methlyxanthine, LB tet + xanthine, and LB tet + theophyyline. I covered the plates in foil and placed in the 37C incubator O/N. Tomorrow I will look for growth to see if the riboswitch can detect 3-methlyxanthine and xanthine.

cadoyle, 11 October 2013

I looked at the plate experiment from yesterday the results are:

Tet +Theophylline = lots of growth

Tet + caffeine = 1 colony

Tet + 3methyl = no growth

Tet + xanthine = no growth

Tet= no growth

It is possible that the concentration of 3-methylxanthine and xanthine needs to be higher in plates than in liquid culture. At .1mM we saw GFP expression in liquid culture but it could vary for tetA. I am not sure if the colony on Tet + caffeine is showing that Tet degraded or if the cells were able to make theophylline? The negative control showed no growth indicating Tet is working. Therefore, I would presume that we have a strain of e.coli that mutated to make theophylline. I will grow up the colony and test it again on plates with caffeine plus tetA to see if can grow.

I took the colony from the plate with caffeine and plated on a Tet, Tet + Theophylline plate to save it and on a Tet + caffeine plate to see if it can really grow. I will leave it over the weekend at room temperature.

Also, we got an email from the lab that is providing caffeine addicted cells. Hi Malcolm,

MTA done! -- We'll send you the guaB + pDCAF3 strain as a stab next week. You'll want to grow it in the minimal media supplemented with caffeine and antibiotic at 30°C as described in the ACS Synthetic Biology paper. We sometimes get inconsistent results after growing without caffeine or at 37°C (likely due to the enzymes from soil not being stable at 37°C or due to mutations). If you have any other questions about the strain or run into problems using it, you can get in touch with Erik Quandt, the graduate student who led the study (who I am CC'ing to correct any details I may have wrong).

Best, --Jeff

It is possible that ecdM8 is unstable at 37C and that is why we don't see growth!

I took the culture from 10-10-13 and plated the cells on Tet, Tet + caffeine and Tet + theophylline and will let grow over the weekend at room temperature.

Lab Presentation for the week File:LabMeeting10-11-13.pptx

cadoyle, 16 October 2013

I examined the plates that grew over fall break. Tet - growth Tet + Theophylline - growth Tet + Caffeine - growth

This indicates that there is contamination or the tetA resistance is gone.

For pictures see lab meeting presentation 10-18-13

MoWest sent data indicating that the concentration of Caffeine should be higher. I will try the experiment again at room temperature but with 1mM Caffeine plates.

File:CDM8 Data.pptx

The results from the colony that grew on caffeine + Tet on 10-11-13: Tet- growth Tet + theophylline-growth Tet=Caffeine-growth

This indicates that the strain is making theophylline but a mutant that evolved to be resistant to Tet.

I realized that J40 is in psB1A2 and not psB1A8 this means that GGA might not work since psB1A2 has bsa I site. I will go ahead and try GGA to see if it will work but will also start working on cloning J40 into psB1A8.

GGA I performed GGA following this protocol [12]

	Neg control (J40)	J40 + J303
dh20	6ul	5ul
J40	1ul	1ul
10X ligation buffer	1ul	1ul
KoAC	1ul	1ul
BsaI	0.5ul	0.5ul
T4 ligase	0.5ul	0.5ul
J303	0.5	1ul
Total	10ul	10ul

PCR Cycles: 20 cycles of 37C for 1min and 16C for 1min hold 22C

Digestion fo J40 (Insert) and J303(has psb1A8)

	J40	J303
DNA	3ul	3ul
dH20	13ul	13ul
10X ligation buffer	2ul	2ul
EcoRI	1ul	1ul
Pst	1ul	1ul
Total	20ul	20ul

Incubated for 3 hours at 37C

I started a 2mlLB = AMp + Chlor culture of J40 + J303 and grew o/n at 37 C shaking.

Also, I edited my proposal: File:DoyleThesisProposal.doc

cadoyle, 17 October 2013

I made a .4% gel with 0.5X TBE and ran the digestion of J303 for 45 minutes. I cut out the band around 2100kb and saved for gel purification.

I plated the culture of J40 and j303 with a sterile loop on Tet, Tet + Theo (0.4mM) and Tet + Caffeine (1mM). I incubated at room temperature o/n

I transformed GGA product following the protocol [13]

I plated on LB + Amp and grew o/n at 37C

cadoyle, 18 October 2013

I looked at the transformation with GGA. There were lots of colonies. I am not sure the transformation worked. I performed colony PCR with 2 controls and 10 experimental following this protocol. [14]

I looked at that experiment with J40 + J303 plated on Tet, Tet + Theo, Tet + Caff. There was no growth on any plates. So, I put the plates in 30C incubator. I will check the plates on Sunday for growth.

I made a 0.4% agarose gel and ran the colony PCR on the gel as well as J404 from digestion.

GGA Results:

Lane 1: MW Lane2-11: samples 1-10 lane 12: control 1 lane 13: control 2

None of colonies contained the insert. The insert should be around 34300kb and the gel shows no bands mainly just primer. This is probably due to the fact that psb1A2 has a bsaI site. I will continue to work on cloning J40 into psb1A8.

Digestions Results:

lane 1 MW Lane 2: J40 digested with Pst and EcoR1

The digestion was successful. There is some DNA that is undigested. Top band. The insert should be about 1300kb and it is. I will cut out the band and gel purify with psb1A8.

Lab Meeting Presentation: File:LabMeeting10-18-13.pptx

cadoyle, 21 October 2013

My objective today is to gel purify, ligate, and transform J40 into psb1A8

Gel Purification: Following this protocol [15]

J40 and psB1A8 gel pieces weighed 0.1g. I added 200ul of NT binding buffer.

Nano Drop: J40: 1.42ng/ul A260: 0.028 A280: -0.012 260/280: -2.23 260/230 -0.15 psB1A8: 1.13ng/ul A260: 0.023 A280: 0.006 260/280: 3.54 260/230 0.05

The numbers are very low. I probably should have digested 3000ng not 1500ng. I will continue with ligation and transformation but the probability the transformation will work is very low.

Ligation: Following this protocol [16] We want 92ng of insert for 50ng of plasmid. The insert is 1300bp and the plasmid is 2100bp. I do not have 92ng of insert of 50ng of plasmid. I will spin down both insert and plasmid and re suspend the plasmid in 2ul of dH20 and the insert in 1ul of dh20.

	Neg control psb1A8 only	J40
dh20	3ul	2ul
J40	0ul	1ul
2X ligation buffer	5ul	5ul
T4 ligase	1ul	1ul
Total	10ul	10ul

Transformation: Following this protocol [17]

10ul of ligation, 50ul of cells, 40ul of SOC and 1ul of .1mM Theophylline

Let the cells sit for 55 minutes than plated on Tet + Theophylline plates and incubated o/n at 37C

Tomorrow I will look fro growth to see if transformation worked!

I took the plates out of the incubator from 10-18-13. There was growth on the negative control so TetA resistance is probably gone. I will remake plates and retry the experiment.

plate 1: TetA Plate2: Tet + 0.1mM Caffeine Lane 3: Tet + 0.4mM Teophylline

cadoyle, 22 October 2013

I checked the transformation. There was three colonies!!!! The transformation was successful. I will grow the colonies in 3mls of LB + AMP and miniprep.

I made a 2ml culture of J40+J303 in LB + chlor + Amp and incubated at 37C

Miniprep of j40 in psb1A8: following this protocol [18]

Nano drop: J40: 1.42ng/ul A260: 0.028 A280: -0.012 260/280: -2.23 260/230 -0.15

Digestion to verify insert:

Digestion fo J40 (Insert) and J303(has psb1A8)

	J40 in 1A8 samples 1,2,3	J303 in 1A8
DNA	3ul	3ul
dH20	13ul	13ul
10X ligation buffer	2ul	2ul
EcoRI	1ul	1ul
Pst	1ul	1ul
Total	20ul	20ul

I incubated at 37C for 1:30min

cadoyle, 23 October 2013

I made a .4% gel and ran the digestions fro yesterday to verify insert size.

Lane 1: MW Lane 2: empty Lane 3 Sample 1 Lane 4: sample 2 lane 5 control plasmid with original insert lane 6:sample 3

There should be 1k bp difference between the original insert and the new insert. It is hard to tell if the insert is correct there is a slight shift up so I will continue with sample 1 in GGA.

I ran an inverse PCR using primers to add BSAI sites.

PCR in preparation for GGA: J110140 Master mix 50ul FWD Primer 1ul REV Primer 1ul DNA 1ul Water 47ul

J119303 Master mix 50ul FWD Primer 1ul REV Primer 1ul DNA 1ul Water 47ul

PCR Cycle for J110140 Step 1: 95° C 5 minutes (denature template) Step 2: 95° C 30 seconds (denature dsDNA) Step 3: 61° C 30 seconds (Tm minus 5 degrees) Step 4: 72° C 3 minutes Step 5: Repeat Steps 2 through 4, 29 more times Step 6: Store at RT°

PCR Cycle for J119303 Step 1: 95° C 5 minutes (denature template) Step 2: 95° C 30 seconds (denature dsDNA) Step 3: 57° C 30 seconds (Tm minus 5 degrees) Step 4: 72° C 1 minutes Step 5: Repeat Steps 2 through 4, 29 more times Step 6: Store at RT°

GGA of J303 into J40: Following this protocol [19]

	Neg control (J40)	J40 + J303
dh20	6ul	5ul
J40	1ul	1ul
10X ligation buffer	1ul	1ul
KoAC	1ul	1ul
BsaI	0.5ul	0.5ul
T4 ligase	0.5ul	0.5ul
J303	0ul	1ul
Total	10ul	10ul

PCR Cycles: 20 cycles of 37C for 1min and 16C for 1min 1 cycle 37C for 15 minutes hold 22C

Transformation: I transformed using the zippy protocol.

10ul of GGA ligation 50ul of cell 40ul of SOC

plated on LB AMP and incubated at 37C o/n. Tomorrow will do colony PCR to verify results.

cadoyle, 23 October 2013

I got growth fro my transformation about a 2:1 ratio between control and experimental.

Colony PCR I performed colony PCR with 2 controls and 10 experimental following this protocol. [20] I used Fwd J40 and Rev J303 Gel of Colony PCR.

I made 0.4% gel and ran the samples Lane1: MW Lane 2-11: samples 1-10 Lane 12: control1 lane 13: control2

The curvature of the band indicates a difference is salt concentration between the TBE in the box and in the gel. Also, the control and the insert did not get amplified. THe PCR product should about 4-5kb and I only amplified for 3 minutes so the product would not have been amplified. I am going to grow the samples on TetA + Theophylline and if they grow than I know the TetA module is there. Then using the primers for ecdm8 (J303) I can see if ecdm8 is present. I going home tomorrow and will be back late Saturday so will start growth then.

cadoyle, 25 October 2013

I looked at the plates that were streaked with ecdm8 and TetA on 10-22-13. There was growth in the Tet + theophylline but not Tet and Tet + caffeine. See lab meeting presentation for pictures. This is probably because the cells have to be producing theophylline before they can grow on Tet so they will get killed before they can mutate to make theophylline and be tetA resistant.

Lab Meeting Presentation: File:LabMeeting10-25-13.pptx

cadoyle, 26 October 2013

Today my objective is to work on fixing the program. I worked from home trying to create loops that will run the program for a given combination of riboswitchs and grab the results and put them into a dictionary.

cadoyle, 27 October 2013

Today I went to the lab and implemented what I work on home yesterday and successfully got the program to take a given riboswitch run it through una-fold, grab the results, and place results less than the free energy of the aptamer in a dictionary with its free energy.

I ran the program for the theophylline aptamer to check the accuracy of the results.

I took the colonies I streaked out to save 10-23-13 from colony PCR and grew on Tet + Theophylline to see what cells have the TetA fitness module present. I grew them o/n at 37C. Tomorrow I will grow up the colonies that grew and then perform PCR.

cadoyle, 28 October 2013

I meet with Dr. Campbell today about the program. I need to implement a ticker to see how long it will take the program to run the combinations. Also, I need to generate a ppt where we can compare the sequences of Riboswitch D and E from Topp et al. that are not published with B and E. This will allow us to look at the results from the program and generate a better idea of what type of riboswitch we need.

Seven out the 10 colonies grew on LB + tet indicating they have the Tet fitness module. I started LB AMP cultures for the colonies that grew on Tet + Theophylline. I grew o/n at 37C.

I made a ppt slide to compare riboswitches and will post on Friday.

cadoyle, 29 October 2013

I went to perform a mp and the cultures were not shaking so the cells did not grow. I started the shaking and will miniprep later. I talked to Dr. Campbell but he said to just do a colony PCR of the colonies instead of mp.

Colony PCR of colonies containing the TetA module looking for ecdm8:

I followed this protocol using the PCR primers for ecdm8 fwd and rev: [21] I ran colony PCR cycle following the cycle on the protocol with 3:30min amplification.

I realized I was randomizing the wrong 8bp so I stopped the program and fixed it to generate random combinations of the 8bp between the RBS and stop codon.

cadoyle, 30 October 2013

I made a 0.4% gel to run the colony PCR product on, but it leaked out of the gel. I remade the gel and it happened again. I remade it a third time with a new gel cassette.

I ran the gel for 30 minutes: Lane 1: MW Lane 2: Space Lane 3: Control Lane 4: 1 lane 5: 3 Lane 6: 4 Lane 7: 5 Lane 8: 6 Lane 9 7 Lane 10: 10

None of the colonies show a band around 3300bp they are identical to the control. There is a lot of primer amplified. I will redo GGA and try redoing the colony PCR with different annealing temperatures.

cadoyle, 31 October 2013

GGA of J303 into J40: Following this protocol [22]

	Neg control (J40)	J40 + J303
dh20	6ul	5ul
J40	1ul	1ul
10X ligation buffer	1ul	1ul
KoAC	1ul	1ul
BsaI	0.5ul	0.5ul
T4 ligase	0.5ul	0.5ul
J303	0ul	1ul
Total	10ul	10ul

PCR Cycles: 20 cycles of 37C for 1min and 16C for 1min 1 cycle 37C for 15 minutes hold 22C

Transformation: I transformed using the zippy protocol.

10ul of GGA ligation 50ul of cell 40ul of SOC

I incubated at 37 for 30 minutes to let the cell recover than plated on LB Tet + Theophylline and incubated at 37C o/n. Tomorrow will do colony PCR to verify results.

cadoyle, 1 November 2013

I compared the sequences of Topp et al (2010) using m-fold to the pictures they provided and found bp-diffrence on riboswitch B (see ppt). Other than that the sequences are the same. When the program is finished I will compare the results and see if we can cut out possible answers. File:Ribocomparrison.pptx

Colony PCR I performed colony PCR with 2 controls and 10 experimental following this protocol. [23] I used Fwd J303 and Rev J303

Gel of Colony PCR.

I made 0.4% gel and ran the samples Lane1: MW Lane 2-6: samples 1-5 Lane 7: control1

I ran the gel for 30 minutes.

Again the PCR product is not amplified and only primers are present. There might be something wrong with the PCR program. It might not be denaturing. Therefore, when I originally performed the iPCR the product the bsaI sites were never added. I will redo iPCR and try again.

cadoyle, 2 November 2013

iPCR fo J40 and J303 to add bsaI sites

	J40	J303
DNA	1ul	1ul
dH20	47ul	47ul
2x Master Mix	50ul	50ul
Primer 1	1ul	1ul
Primer 2	1ul	1ul
Total	100ul	100ul

PCR was run according to this protocol [24] with anneal temperature of 57 for J40 and 64 for J303. The amplication time was 3:30.

After the PCR ran I cleaned up the PCR product. using this protocol [25]

Nanodrop of Cleaned PCR Product: J40: 147ng/ul A260: 2.953 A280: 1.726 260/280: 1.71 260/230 2.35 J40: 395.76ng/ul A260: 7.915 A280:4.332 260/280:1.83 260/230 2.24

cadoyle, 3 November 2013

GGA of J303 into J40: Following this protocol [26]

	Neg control (J40)	J40 + J303
dh20	6ul	5ul
J40	1ul	1ul
10X ligation buffer	1ul	1ul
KoAC	1ul	1ul
BsaI	0.5ul	0.5ul
T4 ligase	0.5ul	0.5ul
J303	0ul	1ul
Total	10ul	10ul

PCR Cycles: 20 cycles of 37C for 1min and 16C for 1min 1 cycle 37C for 15 minutes hold 22C

Transformation: I transformed using the zippy protocol.

10ul of GGA ligation 50ul of cell 40ul of SOC 1ul of 0.1mM Theophylline Let sit for one hour at 37C

plated on LB AMP + Theophylline and incubated at 37C o/n. Tomorrow will do colony PCR to verify results.

cadoyle, 4 November 2013

I checked the transformations and there were no colonies on the control or experimental plate. I emailed campbell about it. It is possible that the PCR Reaction did not work. I a going to run a gel of the clean up PCR product with the mp of the constructs on a gel and see if the PCR worked.

I made a 0.4% gel and ran the PCR product with the mp as a control.

Lane 1: mW Lane 2: J303 mp Lane 3: J303 ipCR Lane 4: J40 mp Lane 5 J40 ipCR

The PCR did not work J303 should have been about 3200 cut in lane 2 and it is smaller than 3200. J404 might have worked in lane 5 up is very fiant. I think I need to use Immersion PCR master mix for amplification of longer sequences. I will re do ipCR and verify pCR worked before moving to GGA.

I made a 3ml LB Amp culture of J303 and grew o/n at 37C shaking.

cadoyle, 5 November 2013

I performed a miniprep on J303 culture using this protocol: [27]

Nano Drop: J303: 101.5ng/ul A260: 2.030 A280: 1.083 260/280: 1.87 260/230 1.95

iPCR fo J40 and J303 to add bsaI sites

	J40	J303
DNA	1ul	1ul
dH20	47ul	47ul
2x Precision Master Mix	50ul	50ul
Primer 1	1ul	1ul
Primer 2	1ul	1ul
Total	100ul	100ul

PCR was run according to this protocol [28] with anneal temperature of 57 for J40 and 64 for J303. The amplication time was 4:00.

I talked to Dr. Campbell and the program is still running. We calculated how long it would take to run 64,500 combinations based off how fast it was doing one every 10secs. It should have been done in 9 hours. Tomorrow I will install it on another computer and see I figure out what is doing and how long it will take.

cadoyle, 6 November 2013

I installed unafold on another computer following these instructions:

1. Direct to folder where the configure program is located 2. run sh ./configure 3. direct to home of user 4. run make 5. sudo make install and insert password 6. You are done!

I tested how long it will take to make 8^4 combinations and realized that it was making 8^8 combinations for the sequences. Therefore, it is making 16,77,216 combinations. I need to rewrite this part. It is a different logic that what we thought we were doing. It is taking a given sequence and randomizing the bases to get all possible combinations. It is not taking the four nucleotides and making every 8bp combinations with them.

Tomorrow I will try to rewrite this part of the code.

cadoyle, 7 November 2013

My objective today is to try to fix the code so it generate the right number of sequences.

I looked at many options but found that this code worked the best:

i = 0 while len(seq) < 65536:

   x = .join(random.choice('ACUG') for _ in range(8))
   if x not in seq:
       seq.append(x)

for line in seq:

   print line

It takes 10 minutes to generate 65,400 combinations. Next I will run through unfold and time how longs it takes so I can estimate how long it will take for all the combinations.

It takes 4sec to run 4^6 combinations.

I am going to run all 65,536 combinations overnight and check the results in the morning.

cadoyle, 8 November 2013

I checked the program and it did not finish. It does not make sense. Maybe it is the way I organized the script. I restarted it and started one for 3-methlyxanthine. I came back at lunch and they were not done. So I decided to just generate the combinations and then run them through unafold. Maybe it will speed up the time. After two hours neither program running was done.

Lab Meeting Presentation: File:LabMeeting11-8-13.pptx

During lab meeting I talked about about how the program was finished when it should have been. This is because I was using the random function and it was trying to figure out the last combination using random combinations. I need to make program that makes all the combinations systematically. I will work on this tomorrow.

cadoyle, 9 November 2013

MY objective today is to try to generate 4^8 combinations of 8bp sequences. I realized that the way the I was thinking about loops was incorrect. Once the loop finishes it retraces it steps generating a new sequence. Below is the code to generate 4^8 sequences.

for a in bases:

   one = a
   for b in bases:
       two = one + b
       for c in bases:
           three = two + c
           for d in bases:
               four = three + c 
               for e in bases:
                   five = four + d
                   print five
                   for f in bases:
                       six = five + f
                       for g in bases:
                           seven = six + g
                           for h in bases:
                               eight = seven + h

It takes 4secs to generate all 64,000 combinations

On Monday I will implement this using una-fold and see how longs it takes to run the whole script.

cadoyle, 11 November 2013

I implemented unafold with the sequence combination generator. It took about 3 hours to generate 16,284 sequences. Based of the literature I will take the lowest 10 sequences and run them through m-fold to compare their folded structures to characteristics of Ribsowtich D and E. From this hopefully we can find reasons to eliminate potential candidates.

I realized that when I ran the program I only got the sequences and need the program to print out the dg for each sequence as well. I will re run the code and generate the dgs for each sequence.

cadoyle, 12 November 2013

I got the results back from the program and wrote a script to pull the dgs from the dictionary.

infile = open("Results11-12-13.txt" , "rU") dgs= [] for line in infile:

   seq, dg = line.split(" ")
   dgs.append(dg)

outfile = open("out.txt", "w") for line in dgs:

   outfile.write(line)

I copied the dgs to an excel file and sorted the sequences by lowest dg.

I took the 10 sequences with the lowest dg and ran them through mfold to generate structures.

File:TheophyllineResults11-11-13.pptx

I taked to dr. Campbell and we realized that lowest dg is not sufficient. We re-read the Lynch et al. paper and realized that what made the riboswitch successful is the difference between the off folded structure and on unfolded structure was less than the absolute value of the dg of the aptamer to bind to theophylline. We realized that the unfolded structure represented how theophylline binds to the aptamer. We looked at the reference for folding the on switch Jensen et al (1994). There are 12 bases where theophylline will bind to the aptamer. Lynch et al. forces the base pairing of these bases and prohibited the base pairings of the rbs using constraints on m-fold. I will try to figure out how they did this and replicate it with Riboswitch D and their sequences.

File:Jension et al 1994.pdf

cadoyle, 14 November 2013

I read the manual on how to use m-fold and determined that to prohibit base pairing of the rbs to the aptamer is how Lynch et al. got the on structure of the riboswitch. Using the input P <starting base pair> 0 <# of base pairs after to stop base pairing> to prohibit binding

I ran the two sequences from lynch et al and verified them as well as Riboswitch D. The difference in the dg was less than 9.5. See lab presentation for images.

I will run the sequences in the program through unafold twice and the second time impose the constraints P 39 0 16. Then I will take the difference of the two dgs and keep only the ones that are less than 9.5.

cadoyle, 14 November 2013

I looked at the results from the program ran last night. There were 13,551 switches that are possible. to narrow down these options I looked to see the range of delta G's are between riboswitch D and riboswitch E of Topp et al (2010). Riboswtich D had a difference of 8.4 and riboswitch E had a difference of 9.4. Therefore, since the delta G of theophylline binding is 9.2 I will keep all the Delta g's that have a difference between 9.1- 8.4. This brought the candidates to 1,554. I need to determine a way to narrow down these candidates. I talked to Dr. Campbell and it seems to be that only difference in delta G is between how the RBS base pairs to the aptamer. In D the rbs base pairs on the 3' side and has 1 free base. In E which is stronger ON and weaker OFF binds on the 5' side and has 2 free bases. I will look to see if that makes a difference. Also, in the 8bp there are two bases that don't bind for D. We wonder if the delta G will change if the bases were different and did base pair. It might be that 6 base pairs that do base pair are essential for the off position. I am going to run the program for 9bp to be randomized since riboswitch D has a 9bp fragment not 8 to compare.

Lab Presentation: File:Labpres11-15-13.pptx

Results with difference in Delta G calculated: File:RESULTS11-15-13.xlsx

cadoyle, 15 November 2013

After lab meeting we realized that we need to see what the dG differences are between A, B, C, D, and E riboswitches before we make the acceptable range of dg differences to be 8.4-9.1 we need look at the function of the other switches and the differences in their dG. I ran all the switches through unafold and calculated their differences. (see lab presentation for this week). From the differences it seems that the acceptable range if 8.4-9.1 is logical based off the higher differences for switches with poor offs and ons.

I also, separated out the switches that are between the 8.4-9.1 range and there are 1,879 switches. I need to figure out a way to see if start codon and RBS base pair to the aptamer.

cadoyle, 16 November 2013

To see if the RBS an start codon base pair to the aptamer I need to figure out how to read the matrixes generated and how they related to the structure. I compared a structure from m-fold to the matrix generate by unafold for ribsowtch D. I determined that column 1 represents the base number and column five shows what base it base pairs to. 0 indicates that it does not base pair. I need to write to generate a way to screen the matrix to see if the start codon and RBS base pair.

I work on the code for about 5 hours and could not get the program to read the file and pull out the right values. I will work on it again tomorrow.

cadoyle, 17 November 2013

I was finally able to get the program to work!!!. I needed to strip the lines first with \n before splitting them.

I ran the program and got 179 sequences base paired to the start codon and that 14 also base paired to the RBS. However, I lost Riboswitch D during the check to base pair tothe Start codon. This should not happen because D does bas pair to the RBS and Start codon.

cadoyle, 18 November 2013

I checked out the matrix and there was a difference in the matrix numbers between the ones generate by unafold and the ones generate m-fold. This is why Riboswtich D gets removed. I looked una-fold information and it says that m-fold and una-fold have different algorithms therefore, this could explain the differences in matrix and structure alignments. I talked to Dr. Campbell and we are going to run D, and E and 8.1 and 8.2 with known structures and function data to see if we can see how m-fold differs from una-fold.

cadoyle, 19 November 2013

I ran all switches D, E, 8.1, and 8.2 through unafold and m-fold. I found that the number of possible structures varied. However, only E differed in the actual values. I think that how I was running the program only looked at one file. If only one file was being looked at it could be the file of the structure that is not the one use by Topp et al. I will try to edit the program. I tried editing the program but the program cannot write to the file. I tried debuging for two hours and cannot get it to work. The file is made but nothing is written to it. I checked the permission they are fine, everything is saved in the same place, and the wing version works. I emailed Dr. Heyer to get her help.

cadoyle, 20 November 2013

Dr. Heyer emailed me back and suggested checking to see if the file was saved properly or if the premissions were incorrect. I did and they were fine. I tried just opening a file and writing hello and it does not work. I can run una-fold from command line but not from python. I tried a bunch of things for about two hours but nothing worked.

cadoyle, 21 November 2013

I worked on the lab presentation today and continued to debugg. I updated Dr. Heyer and seems that maybe I need to check what happens at command line. I f switched back to Dr. Campbell's username and the program worked. I realized that a file must already be present for unafold to work. I am not sure why but there is already a file it works. I realized also, that the reason that Riboswtich D was thrown out was because Topp et al had a 9bp spacing instead of 8 like I was doing. Therefore the one extra base pair changed the base pairing. I am going to run the program to generate combinations for 9bp randomized sequence.

cadoyle, 22 November 2013

Sadly the program ran but there was one variable undefined so it stopped. I will re start the program and it should be done in a couple of hours. Then hopefully I will have time to run through and gather info! I going to check out Topp et al paper to see why they switched to 9bp and not 8bp. I looked back at the paper and found that the 8bp I am randomizing were randominzed but also 12 base pairs were randomized as well. The 12 base pairs include the RBS. Therefore, it is 9bp that are randomized but a typo in the Topp et al paper that shows 9 bp when it mean to say 12 and 8 that were randomized. Maybe I should be randomizing two sequences. See lab presentation for data. That paper is Lynch and Gallivan (2009) File:Gkn923.pdf

The results came back from 9bp but I didn't change the base pairings. I have to re run it. It should be done before lab meeting but will not have tim to analyze the data. I will have wait till I get home.

Lab Presentation: File:Labpresetnation11-22.pptx

cadoyle, 23 November 2013

I took the computer home and got it set up. Before I left to go home the program finished. I started analyzing the data and realized that riboswitch D was not present. I found a spacing error in the loop where the riboswitches were being written to a new file. I re-started the program. It takes about 12 hours so will analyze results tomorrow. I also tried writing the switches that had a difference in delta G less than -9.2 to a file.

cadoyle, 24 November 2013

The program had a buffer object error when trying to write the switches that had a difference in delta G less than -9.2 to a file. I will re run the program and just print the switches to output box. I started finishing writing my methods and making figures for the thesis paper.

cadoyle, 25 November 2013

The program worked. I analyzed the data but Riboswith D was not present. Dr. Heyer had suggested letting the program rest each time it went through Unafold. I added two 1sec breaks, each time after it runs through unafold. Hopefully this will work.

cadoyle, 26 November 2013

The program is still running. I sent Dr. Campbell email:

I figured out that the reason riboswitch D was being removed from the pipeline was because we were randomizing 8bp and it seemed that Topp et al for riboswitch D was randomizing 9bp. Therefore, I ran 9bp through the program. When I did this riboswtich D was still removed from the pipeline. Dr. Heyer had suggested that I let the program rest after each calculation. I am running that now it is taking a very long time. However, I ran into an error where the program is not being called every time I call it when I check the base pairing. Possibly this could cause an error with Riboswitch D not being present? I have checked the program and cannot figure out why this occurs. I am going to email Dr. Heyer about it.

Ideas:

1. Stick to randomizing 8bp but add the bp that is missing into the set of bases that are in every switch and see if this fixes the problem
2. See if the reason why riboswitch D is removed is because unafold is not working properly
3. I am attaching the paper I found. In the paper they took the 8.1 clone from Lynch et al 2007 with the set 8bp they found and randomized 11 bases that included the RBS. They found the RBS that worked the best was UAAGG. Then they found two bases after the RBS UA and four before CUGC to make ribosiwtch D. We could keep the set RBS and randomize all three randomized sections or just the 8bp like we had planned. 

cadoyle, 27 November 2013

I stopped the program because it was still running. I think that letting the program pause is just making the program run slower like Dr. Campbell had suggested. I tried to figure out why the program was not being called each time. I took the script and cut the systematic combinations to only two bases. Then I ran switches through the base checker program but it did not write the switches to the file. I renamed the file and it worked. Therefore, each time you run the program you have to rename the file being made or the program gets confused with opening and closing the same file because the fie is not removed. This is probably why Riboswitch D is not there I ran the program through with just 2bp and added riboswitch D and it was kept the whole time. I am going to rerun the program for 9bp and it should be done by 4pm. If all goes well should know if everything is working properly and we can start looking at other apatmers.

cadoyle, 29 November 2013

The program ran but Riboswitch D was not generated during the randomization process. This means that opening and closing the files does not effect riboswitch being an output, but does for checking the bases. Therefore, I did the following tests: 1. Is the program generating the right number of riboswitch sequences? It generated 262,075 when it should have generated 262,144 2. Is it generating 262,144 randomized sequences? It generated 262,144 when it should be generating 262,144 (but D's randomized sequence is not present) 3. Is D present with 4^8 combinations are generated? Yes it is, but it was present 4 times 4. The actually number of sequences being generated that are unique with 4^8 combinations are 16384 5. Therefore, why in number 1 there are less sequences is because I have a parameter that if the sequence is already present do not add it to the list.

I need to figure out how to generate all the combinations without getting repeats. Before, when I was generating the different combinations I was checking the number made and did not check to see if there was repeats. On the bright side if we can generate riboswitch D it is kept through the whole program, which means the program works past the first step.

I started going through the loops one by one. I copied the loops one by one to a new script and ran the loops to see what is being generated. Loops 1-3 worked as expected and I got the right numbers. When I did loop four I got the right number but repeat switches. I looked at the loop from and noticed that I did not replace the variable in the loop. I had c (underlined) instead of D. This is why riboswitch D never got generated. I fixed the script and restarted the program.

for a in bases:

   one = a
   for b in bases:
       two = one + b
       for c in bases:
           three = two + c
           for d in bases:
               four = three + c 
               for e in bases:
                   five = four + e
                   for f in bases:
                       six = five + f
                       for g in bases:
                           seven = six + g
                           for h in bases:
                               eight = seven + h
                               for k in bases:
                                   nine = eight + k
                                   switch = aptseq + "cugcu" + rbs + nine + "aug" # add final iteration to the rest of the riboswitch

cadoyle, 30 November 2013

The program ran and Riboswitch D was there!!!!!! Yay it worked. However, I think there are so many switches the did not all fit in the box. I will re-run the program and print to a file just to make sure I have all the switches.

cadoyle, 1 December 2013

The program finished, but I did not put a space between the switches so I cannot analyze. I re ran the program and added "\n" to each line. The program had a buffer object error where you can not a string and tuple. I fixed the problem but just writing them all in the same file without spaces. I will try to figure out how split each one apart. I re ran the program and just printed the switches again.

cadoyle, 2 December 2013

Riboswitch D was present but it had the wrong dg it had -18.5 not 22.1. I ran just dg alone and it worked. I tired coping a few switches to a file and than read the file and I got the right delta G. Therefore I decided to generate the switches and write to a file than and then read the file line by line. It worked I got the right delta G. I will do this with all the switches. I will have a script generate all the switches print to a file and then get unafold to read that file.

cadoyle, 3 December 2013

The program ran but the delta G was still wrong. When I took a couple from the file and ran them by themselves. 25 above and below riboswtich D in the file it worked. There must be some character that is present when you copy versus write to a file. Dr. Campbell suggested writing a program that will cut those 51 switches and write to a file and than compare the numbers to the copied file to show that there is an error in the spacing. I did try comparing to copy between an excel file and text file and there is a difference in DG, further suggesting there is hidden character.

cadoyle, 4 December 2013

Before doing what Dr. Campbell suggested I just ran a file with a copied Riboswtich D from a script that generated the all the switches file through the script I have been using it worked fine. When I ran the 51 switches again it did not work. Therefore, I wrote a code to find hidden characters. I found that are carriage returns in the file where the riboswtiches are written but not in the file where the switches were copied.

This is what I send Dr. Campbell it summaries everything:

Before doing what we had talked about in our meeting I tired running just riboswitch D alone in a file by itself, where unafold found and opened the file. I got the right delta G. Then when I used the script I had been using to run the same file I got the wrong delta G. The script I am using opens a file, reads  it and takes each line or each riboswitch in the file and writes just that one switch to a new file, which unafold finds and opens. So, I figured there must be something different about a file when you write text to it. I wrote a script that checks for hidden characters and found that when my program wrote to a file a carriage return was present but  if a switch is copied and pasted into a file the carriage return is not present. 

cadoyle, 5 December 2013

I read online that if you write wb when you create a file open("filename", wb) instead of w that extra characters are removed. I did this and it worked the carriage return was not present. I ran the script but still got the wrong delta G. I noticed there was a weird space in the output. I printed line by line and found that when I split the lines the was a blank line. I removed the line hoping that might fix the problem but it does not work. I still get the wrong answers. I started playing with the number of switches in a file and the delta G of riboswtich D changed depending on how many switches. I didn't have time to finish but will pick up here tomorrow.

cadoyle, 6 December 2013

For some reason running just Riboswitch D did not work. When I added one before it worked, but the first switch in the file is not being read. I know that it writes it to a file but it does not open it dg file. I am not sure or how to fix it. I tired placing multiple riboswitches in a file but only one was read. Maybe it is not reading on all the switches. I am not sure. I printed each line in the program but could not figure out what was going on.

I will continue to work on this over break and also finish my draft of my thesis

File:Labpresentation12-6-13.pptx

cadoyle, 2 January 2014

Today I send in my thesis to Dr. Campbell and will start working on developing aptamers for 3-methylxanthine and caffeine. I inputted the 3-methylxanthine aptamer into the program to generate potential riboswitches. However, I needed to calculate the binding energy for the 3-methyxanthine aptamer. I looked at the paper Soukop et al 2000 but there was no binding energy reported. Therefore, I went back to the Lynch et al paper that talked about binding energy to see how they calculated it. I found that that used the kd reported by Jenison et al 1994 to calculate the binding energy of the theophylline aptamer. I looked up how to calculate the binding energy from the kD and found that the equation is: Delta G = RTln(kd). The R is 1.987X10^-3. I checked the equation by plugging in the kd reported from Jenision to see if I got the same value as Lynch et al 2007 reported. I did therefore, I calculated that the binding energy for the 3-methylxanthine aptamer is -7.05 and for the caffeine aptamer is -4.06. I ran the program for 3-methylxanthine.

Thesis: File:DoyleTheisFall2013.docx

cadoyle, 3 January 2014

The program finished running but I forgot to change the values for the ON state of the riboswitch. Therefore, the differences were wrong. I restarted the program and inserted 26 0 22 for prohibiting binding.

cadoyle, 4 January 2014

The program finished and ran the program for caffeine inhibiting binding by inserting 100 0 22.

cadoyle, 5 January 2014

The program finished and Dr. C sent me an email about the results of the paper. He suggested that we needed to minimize the number of possibilities. I wrote back: I agree that we need to minimize the guessing as well out of the 713 switches. The altered 9 nucleotide sequences from B, C and D are in the 713. I use mFold to view each structure I generated that has the altered 9 nucleotide sequence from B, C, and D. I noticed two things 1) the first two nucleotides of each altered nine nucleotide sequence do not base pair. 2) Each sequence contained an A where the nine nucleotide sequence does not pair. B: CA C: AC D: AA

When the two nucleotides do not pair the riboswitch takes on a particular structure that might be favorable due to thermodynamic stability and folding.

On the 713 switches I ran a filter to look for sequences where the first two nucleotides did not pair to the aptamer this took the number of switches down to 74.

The two bases for riboswitch D that do not base pair are As. I started thinking if GC content played a role. The lower the GC content the lower the melting temperature is decreasing the binding energy.

Out of the 74 switches 10 switches have AA that do not bind to the aptamer for the first two bases of the altered 9 nucleotide sequence.

I am not sure if the last one is a reasonable filter. Let me know what you think. I have already ran caffeine and 3-methylxanthine through the longest filter and within a hour I could run these new filters and see what we get.

Results for theophylline Riboswitches:

File:Results12-19-13.xlsx

cadoyle, 8 January 2014

I ran the caffeine switches through grab.py and looking from ones with a difference in free energy between 9.1-8.4 kcal/mol (-29.3→ -30). I took the switches and ran it through basechecker.py and found switches where the RBS and start codon pair the aptamer. Also, I checked that two bases of the 9 nucleotide sequence do not pair to the aptamer. I found 18 that the two bases are As. I checked the structures using mFold but the start codon does not pair to the aptamer. I will have to re-run the program. I must have inputted the wrong numbers in the program.

cadoyle, 9 January 2014

The switches for 3-methylxanthine finished running through the first filter. I copied them and saved as 3-methylswitches.txt. I ran through grab.py looking from ones with a difference in free energy between 9.1-8.4 kcal/mol (-16.7→ -16). There were no switches that passed the test. I am going to go ahead and run the switches through the rest of the filters. I might be that filter is a bad choice.

I started to re-run the caffeine switches since yesterday I found that the 18 switches I filtered down to the start codon did not base pair. I must have made a mistake. I checked the pairing for the start codon between 1. 119 and != 0 2. 121 and != 0 3. 120 and != 0. I checked pairing for RBS 1. 108 and != 0 2. 107 and != 0 3. 106 and != 0 4. 105 and != 0. For the two bases of the 9 altered sequence 1. 110 == 0 2. 111 == 0. Then I looked for AA for the first two bases of the nine nucleotide sequence and got 17 results.

cadoyle, 10 January 2014

I checked the pairing between the start codon in aptamer for the theophylline switches by inputting: 1. 47 and != 0 2. 46 and != 0 3. 45 and != 0. Then to check pairing between RBS and aptamer I checked 1. 34 and != 0 2. 33 and != 0 3. 32 and != 0, 4. 31 and != 0. For the two bases of the 9 altered sequence 1. 35 == 0 2. 36 == 0. Then I looked for AA for the first two bases of the nine nucleotide sequence and got 10686 results. I might need to find a way to narrow the range between a difference of 9.1-8.4 somehow to decrease the number of results.

cadoyle, 12 January 2014

I sent Dr. Campbell the results I had for everything except 3-methylxanthine. I think after getting the results yesterday I need to find a way to filter them down with the difference in delta G’s. Dr. Campbell looked at my results and asked that I can have ClustalW make the phylogenetic trees. That might make it easier for us to group them into families. We might make 4 riboswitches with each riboswitch having a couple variable bases. That would make it easier to screen the outcomes.

Caffeine Riboswitch Results: File:Caffeineriboswitches.xlsx

I put the 8 sequences for theophylline into clustalW and got the guide tree see below:

1. ggugauaccagcaucgucuugaugcccuuggcagcacccugcuaagguAACGUCCAGaug

2. ggugauaccagcaucgucuugaugcccuuggcagcacccugcuaagguAACGUCUAGaug

3. ggugauaccagcaucgucuugaugcccuuggcagcacccugcuaagguAACGUCAUGaug

4. ggugauaccagcaucgucuugaugcccuuggcagcacccugcuaagguAACAUGAGGaug

5. ggugauaccagcaucgucuugaugcccuuggcagcacccugcuaagguAACAUCUGGaug

6. ggugauaccagcaucgucuugaugcccuuggcagcacccugcuaagguAACAGCAAGaug

7. ggugauaccagcaucgucuugaugcccuuggcagcacccugcuaagguAACAACAAGaug

8. ggugauaccagcaucgucuugaugcccuuggcagcacccugcuaagguAACGUGAAGaug

Phylogenetic Tree: File:Phylogenetictreetheophylline.png

There are four groups 1) 1, 2 2) 8,3 3) 6,7 4), 4,5. 3 and 8 are outliers. Group 1: 1 and 2 differ by one base 1. ggugauaccagcaucgucuugaugcccuuggcagcacccugcuaagguAACGUCCAGaug

2. ggugauaccagcaucgucuugaugcccuuggcagcacccugcuaagguAACGUCUAGaug Group 2: 3 and 8 differ by one base 3. ggugauaccagcaucgucuugaugcccuuggcagcacccugcuaagguAACGUCAUGaug 8. ggugauaccagcaucgucuugaugcccuuggcagcacccugcuaagguAACGUGAAGaug Group 3: 6 and 7 differ by one base 6. ggugauaccagcaucgucuugaugcccuuggcagcacccugcuaagguAACAGCAAGaug

7. ggugauaccagcaucgucuugaugcccuuggcagcacccugcuaagguAACAACAAGaug

Group 4: 4 and 5 differ by two bases 4. ggugauaccagcaucgucuugaugcccuuggcagcacccugcuaagguAACAUGAGGaug

5. ggugauaccagcaucgucuugaugcccuuggcagcacccugcuaagguAACAUCUGGaug

Group I put the 17 sequences of caffeine into clustalW and the guide and phylogenetic Tree: Guide Tree:

Phylogenetic Tree:

Caffeine Aptamers (17) 1. ggauguccagucgcuugcaaugcccuuuuagacccugaugaggaucaucggacuuuguccuguggaguaagaucgcgaaacggugaaagccguaggucucugcuaagguAAUAGUCAGaug

2. ggauguccagucgcuugcaaugcccuuuuagacccugaugaggaucaucggacuuuguccuguggaguaagaucgcgaaacggugaaagccguaggucucugcuaagguAAACGCCCGaug

3. ggauguccagucgcuugcaaugcccuuuuagacccugaugaggaucaucggacuuuguccuguggaguaagaucgcgaaacggugaaagccguaggucucugcuaagguAAAGGAUCUaug

4. ggauguccagucgcuugcaaugcccuuuuagacccugaugaggaucaucggacuuuguccuguggaguaagaucgcgaaacggugaaagccguaggucucugcuaagguAAUAUGCGGaug

5. ggauguccagucgcuugcaaugcccuuuuagacccugaugaggaucaucggacuuuguccuguggaguaagaucgcgaaacggugaaagccguaggucucugcuaagguAAGGUGCCCaug

6. ggauguccagucgcuugcaaugcccuuuuagacccugaugaggaucaucggacuuuguccuguggaguaagaucgcgaaacggugaaagccguaggucucugcuaagguAAGAUCCAGaug

7. ggauguccagucgcuugcaaugcccuuuuagacccugaugaggaucaucggacuuuguccuguggaguaagaucgcgaaacggugaaagccguaggucucugcuaagguAACAGCUCGaug

8. ggauguccagucgcuugcaaugcccuuuuagacccugaugaggaucaucggacuuuguccuguggaguaagaucgcgaaacggugaaagccguaggucucugcuaagguAACAUACCGaug

9. ggauguccagucgcuugcaaugcccuuuuagacccugaugaggaucaucggacuuuguccuguggaguaagaucgcgaaacggugaaagccguaggucucugcuaagguAAGGUGUAGaug

10. ggauguccagucgcuugcaaugcccuuuuagacccugaugaggaucaucggacuuuguccuguggaguaagaucgcgaaacggugaaagccguaggucucugcuaagguAAAGGUGUAaug

11. ggauguccagucgcuugcaaugcccuuuuagacccugaugaggaucaucggacuuuguccuguggaguaagaucgcgaaacggugaaagccguaggucucugcuaagguAAUACGCCGaug

12. ggauguccagucgcuugcaaugcccuuuuagacccugaugaggaucaucggacuuuguccuguggaguaagaucgcgaaacggugaaagccguaggucucugcuaagguAAUUUACCGaug

13. gauguccagucgcuugcaaugcccuuuuagacccugaugaggaucaucggacuuuguccuguggaguaagaucgcgaaacggugaaagccguaggucucugcuaagguAAAGUGUAGaug

14. ggauguccagucgcuugcaaugcccuuuuagacccugaugaggaucaucggacuuuguccuguggaguaagaucgcgaaacggugaaagccguaggucucugcuaagguAAUGGUUUGaug

15. ggauguccagucgcuugcaaugcccuuuuagacccugaugaggaucaucggacuuuguccuguggaguaagaucgcgaaacggugaaagccguaggucucugcuaagguAAUAGUCCCaug

16. ggauguccagucgcuugcaaugcccuuuuagacccugaugaggaucaucggacuuuguccuguggaguaagaucgcgaaacggugaaagccguaggucucugcuaagguAAAGCCUAGaug

17. ggauguccagucgcuugcaaugcccuuuuagacccugaugaggaucaucggacuuuguccuguggaguaagaucgcgaaacggugaaagccguaggucucugcuaagguAAUAGUUUGaug

There are seven groups: 1) 2,7 2) 3,10,14,17 3) 9,13,16 4) 1,15 5) 4, 11 6) 5,6 7) 8, 12.

Group 1: 2 and 7 differ by two bases 2. ggauguccagucgcuugcaaugcccuuuuagacccugaugaggaucaucggacuuuguccuguggaguaagaucgcgaaacggugaaagccguaggucucugcuaagguAAACGCCCGaug 7. ggauguccagucgcuugcaaugcccuuuuagacccugaugaggaucaucggacuuuguccuguggaguaagaucgcgaaacggugaaagccguaggucucugcuaagguAACAGCUCGaug

Group 2: 3. ggauguccagucgcuugcaaugcccuuuuagacccugaugaggaucaucggacuuuguccuguggaguaagaucgcgaaacggugaaagccguaggucucugcuaagguAAAGGAUCUaug 10 ggauguccagucgcuugcaaugcccuuuuagacccugaugaggaucaucggacuuuguccuguggaguaagaucgcgaaacggugaaagccguaggucucugcuaagguAAAGGUGUAaug

Group 3 14. ggauguccagucgcuugcaaugcccuuuuagacccugaugaggaucaucggacuuuguccuguggaguaagaucgcgaaacggugaaagccguaggucucugcuaagguAAUGGUUUGaug 17. ggauguccagucgcuugcaaugcccuuuuagacccugaugaggaucaucggacuuuguccuguggaguaagaucgcgaaacggugaaagccguaggucucugcuaagguAAUAGUUUGaug

Group 3 9 ggauguccagucgcuugcaaugcccuuuuagacccugaugaggaucaucggacuuuguccuguggaguaagaucgcgaaacggugaaagccguaggucucugcuaagguAAGGUGUAGaug 13 gauguccagucgcuugcaaugcccuuuuagacccugaugaggaucaucggacuuuguccuguggaguaagaucgcgaaacggugaaagccguaggucucugcuaagguAAAGUGUAGaug 16 ggauguccagucgcuugcaaugcccuuuuagacccugaugaggaucaucggacuuuguccuguggaguaagaucgcgaaacggugaaagccguaggucucugcuaagguAAAGCCUAGaug

Group 4: 1. ggauguccagucgcuugcaaugcccuuuuagacccugaugaggaucaucggacuuuguccuguggaguaagaucgcgaaacggugaaagccguaggucucugcuaagguAAUAGUCAGaug

15 ggauguccagucgcuugcaaugcccuuuuagacccugaugaggaucaucggacuuuguccuguggaguaagaucgcgaaacggugaaagccguaggucucugcuaagguAAUAGUCCCaug

Group 5: 4 ggauguccagucgcuugcaaugcccuuuuagacccugaugaggaucaucggacuuuguccuguggaguaagaucgcgaaacggugaaagccguaggucucugcuaagguAAUAUGCGGaug 11 ggauguccagucgcuugcaaugcccuuuuagacccugaugaggaucaucggacuuuguccuguggaguaagaucgcgaaacggugaaagccguaggucucugcuaagguAAUACGCCGaug

Group 6: 5 ggauguccagucgcuugcaaugcccuuuuagacccugaugaggaucaucggacuuuguccuguggaguaagaucgcgaaacggugaaagccguaggucucugcuaagguAAGGUGCCCaug

6. ggauguccagucgcuugcaaugcccuuuuagacccugaugaggaucaucggacuuuguccuguggaguaagaucgcgaaacggugaaagccguaggucucugcuaagguAAGAUCCAGaug

7. ggauguccagucgcuugcaaugcccuuuuagacccugaugaggaucaucggacuuuguccuguggaguaagaucgcgaaacggugaaagccguaggucucugcuaagguAACAGCUCGaug

Group 7: 8. ggauguccagucgcuugcaaugcccuuuuagacccugaugaggaucaucggacuuuguccuguggaguaagaucgcgaaacggugaaagccguaggucucugcuaagguAACAUACCGaug 12. ggauguccagucgcuugcaaugcccuuuuagacccugaugaggaucaucggacuuuguccuguggaguaagaucgcgaaacggugaaagccguaggucucugcuaagguAAUUUACCGaug

cadoyle, 13 January 2014

I talked to Dr. Campbell and I need to come up with a way to clone all 17 switches for caffeine. We decided that the best way to test theophylline riboswitches would be to clone the one most different (#1) from riboswitch D to show how my model works. I will make a G-block design of that and also think about a way to clone the rest.

Below is the design for cloning the synthesized riboswitch for theophylline into the reporter construct.

Riboswitch to by synthesized: gcatggtctcaaaatcataaaaaatttatttgctttgtgagcggataacaattataatagattcaattgtgagcggataacaattactagagatacgactcactataggtaccggtgataccagcatcgtcttgatgcccttggcagcaccctgctaaggtaacgtccagatgcctctggtacg

Navy- GCAT Orange- BsaI site Light purple – 1 nucleotide spacer Red – GGA prefix Green- promoter Dark purple is KPN1 site Light blue – Riboswitch

To design the riboswitch and primers for GGA I used the primer time primer designer tool (http://gcat.davidson.edu/SynBio13/primer/) . I inputted J10079 plus the sequence for pSB1A8 plasmid in the plasmid box. I inputted the riboswitch sequence in the insert box. I set the Primer length 20 Starting location of cut 27 Ending location of cut 165 Stick ends on insert.

PCR Product will be 2,984bp.

J100079 in pSB1A8 aaatcataaaaaatttatttgctttgtgagcggataacaattataatagattcaattgtgagcggataacaattactagagatacgactcactataggtaccggtgataccagcatcgtcttgatgcccttggcagcaccctgctaaggtaacaacaagatgcgtaaaggcgaagagctgttcaccggtgttgttccgattctggttgaactggatggtgatgttaatggccacaaattttcagttcgtggtgaaggcgagggtgatgcaaccaatggtaaactgaccctgaaatttatctgtaccaccggcaaactgccggttccgtggccgaccctggttaccaccctgacctatggtgttcagtgttttgcacgttatccggatcatatgaaacagcacgattttttcaaaagcgcaatgccggaaggttatgttcaagaacgtaccatctcctttaaagatgatggcacctataaaacccgtgccgaagttaaatttgaaggtgacaccctggtgaatcgtattgagctgaaaggcatcgatttcaaagaggatggtaatatcctgggccataaactggaatataacttcaatagccacaatgtgtatatcaccgcagacaaacagaaaaacggcattaaagccaactttaagattcgccataatgtggaagatggtagcgtgcagctggcagatcattatcagcagaataccccgattggtgatggtccggttctgctgccggataatcactatctgagcacccagagcgttctgagcaaagatccgaatgaaaaacgtgatcacatggtgctgctggaatttgttaccgcagcaggtattacccatggtatggatgaactgtacaaatgatgatactagtagcggccgctgcagtataaacgcagaaaggcccacccgaaggtgagccagtgtgactctagtagagagcgttcaccgacaaacaacagataaaacgaaaggcccagtctttcgactgagcctttcgttttatttgatgcctgggcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgggacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggcagaatttcagataaaaaaaatgatttctgcgccgcaaaccccgcccctgacagggcggggtttcgccgcgaattcgcggccgcttctagaggcatacagtcga

Green is GFP and Red is plasmid.

Insert: aaatcataaaaaatttatttgctttgtgagcggataacaattataatagattcaattgtgagcggataacaattactagagatacgactcactataggtaccggtgataccagcatcgtcttgatgcccttggcagcaccctgctaaggtaacgtccagatg

J100079 Forward GCAT GGTCTC T GATG AAAGGCGAAGAGCTGTTCAC

BSAI 1bp stick 20mer GFP
         spacer end

>>>Melting temp: 56.2 °C >>>Self dimer ΔG: -6.3 kcal.mole-1 >>>Hairpin ΔG: -3.1 kcal.mole-1

J100079 Reverse GCAT GGTCTC G ATTT ACAAAGCAAATAAATTTTTTATGATTTCT BSAI 1bp stick 20mer of old riboswitch

         spacer end

>>>Melting temp: 53.2 °C >>>Self dimer ΔG: -9.3 kcal.mole-1 >>>Hairpin ΔG: -3.3 kcal.mole-1

Insert Primer 1 – Insert_forward >>>GCAT GGTCTC A AAATCATAAAAAATTTATTTGCTTTGTG >>>Melting temp: 52.6 °C >>> Self dimer ΔG: -9.3 kcal.mole-1 >>>Hairpin ΔG: -4.3 kcal.mole-1

Insert Primer 2 – Insert_reverse >>>GCAT GGTCTC C CATCTGGACGTTACCTTAGC >>>Melting temp: 56.2 °C >>>Self dimer ΔG: -6.5 kcal.mole-1 >>>Hairpin ΔG: -1.6 kcal.mole-1

cadoyle, 14 January 2014

My objective today is to design a way to clone the caffeine riboswitches. I was thinking that similar to how I planned on cloning the theophylline riboswitch into the reporter construct I could design the riboswitch to synthesized. Then I would design unique primers to change the individual bases that differ between the putative riboswitches. Then you could do one iPCR reaction for each riboswitch and preform GGA. See Design Below:

I talked to Dr. Campbell and my design will work expect a faster way to screen would be to add a PST site so if GGA worked than the riboswitch will not have a PST site and we do not need to send for sequencing every time. I am going to design both the caffeine and theophylline riboswitch to be synthesized and then start working on primers for 17 caffeine switches. I fixed the theophylline primers see above (1-13-14) and riboswitch to be sequenced.

cadoyle, 15-16 January 2014

Today I am going make a presentation to show Dr. Campbell see Riboswitch Design Parts.pptx and finish the caffeine riboswitch with a PST site.

Caffeine 1 Gcatggtctctaaatcataaaaaatttatttgctttgtgagcggataacaattataatagattcaattgtgagcggataacaattactagagatacgactcactataggtaccggatgtccagtcgcttgcaatgcccttttagaccctgatgaggatcatcggactttgtcctgtggagtaagatcgcgaaacggtgaaagccgtaggtctctgctaaggtaatagtctgcagtctctggtacg

Navy- GCAT Orange- BsaI site Light purple – 1 nucleotide spacer Red – GGA prefix Green- promoter Dark purple is KPN1 site Light blue – Riboswitch Pink - PST1 site

Dr. Campbell and I meet and went over the riboswitches we went ahead and ordered them from IDT. We changed the caffeine one so the PST site was not the sticky end. I will make primers to fix the cloning into the reporter plasmid. Theophylline Riboswitch to by synthesized: gcatggtctcaaaatcataaaaaatttatttgctttgtgagcggataacaattataatagattcaattgtgagcggataacaattactagagatacgactcactataggtaccggtgataccagcatcgtcttgatgcccttggcagcaccctgctaaggtaacgtccagatgcctctggtacg

Caffeine Riboswitch gcatggtctctaaataaatcataaaaaatttatttgctttgtgagcggataacaattataatagattcaattgtgagcggataacaattactagagatacgactcactataggtaccggatgtccagtcgcttgcaatgcccttttagaccctgatgaggatcatcggactttgtcctgtggagtaagatcgcgaaacggtgaaagccgtaggtctctgctaaggtaatagtcagatgctgcaggatgtctctggtacg

To design primers for caffeine switches:

J100079- G Forward GCAT GGTCTC T GATG AAAGGCGAAGAGCTGTTCAC

BSAI 1bp stick 20mer GFP
         spacer end

>>>Melting temp: 56.2 °C >>>Self dimer ΔG: -6.3 kcal.mole-1 >>>Hairpin ΔG: -3.1 kcal.mole-1

J100079- U Forward >>>GCAT GGTCTC T TATG AAAGGCGAAGAGCTGTTCAC >>>Melting temp: 56.2 °C >>>Self dimer ΔG: -6.7 kcal.mole-1 >>>Hairpin ΔG: -3.1 kcal.mole-1

J100079- C Forward >>>GCAT GGTCTC T CATG AAAGGCGAAGAGCTGTTCAC >>>Melting temp: 56.2 °C >>>Self dimer ΔG: -8.5 kcal.mole-1 >>>Hairpin ΔG: -3.1 kcal.mole-1

J100079-A Forward >>>GCAT GGTCTC T AATG AAAGGCGAAGAGCTGTTCAC >>>Melting temp: 56.2 °C >>>Self dimer ΔG: -6.3 kcal.mole-1 >>>Hairpin ΔG: -3.1 kcal.mole-1

J100079 Reverse GCAT GGTCTC G ATTT ACAAAGCAAATAAATTTTTTATGATTTC BSAI 1bp stick 20mer of old riboswitch

         spacer end

Caffeine Forward >>>GCAT GGTCTC C AAATCATAAAAAATTTATTTGCTTTGTG >>>Melting temp: 52.6 °C >>> Self dimer ΔG: -9.3 kcal.mole-1 >>>Hairpin ΔG: -4.9 kcal.mole-1

Caffeine 1 forward aaatcataaaaaatttatttgctttgtgagcggataacaattataatagattcaattgtgagcggataacaattactagagatacgactcactataggtaccggauguccagucgcuugcaaugcccuuuuagacccugaugaggaucaucggacuuuguccuguggaguaagaucgcgaaacggugaaagccguaggucucugcuaagguAAUAGUCAGaug

Caffeine 1 Reverse GCAT GGTCTC C CATCTGACTATTACCTTAGCA >>>Melting temp: 52.9 °C >>>Self dimer ΔG: -6.5 kcal.mole-1 >>>Hairpin ΔG: -2.6 kcal.mole-1

Caffeine 2 aaatcataaaaaatttatttgctttgtgagcggataacaattataatagattcaattgtgagcggataacaattactagagatacgactcactataggtaccggatgtccagtcgcttgcaatgcccttttagaccctgatgaggatcatcggactttgtcctgtggagtaagatcgcgaaacggtgaaagccgtaggtctctgctaaggtAAtAGtCAGatg

Caffeine 2 Reverse GCAT GGTCTC G CATCTGACTATTACCTTAGCA >>>Melting temp: 52.9 °C >>>Self dimer ΔG: -5.4 kcal.mole-1 >>>Hairpin ΔG: -0.5 kcal.mole-1

Caffeine 3 aaatcataaaaaatttatttgctttgtgagcggataacaattataatagattcaattgtgagcggataacaattactagagatacgactcactataggtaccggatgtccagtcgcttgcaatgcccttttagaccctgatgaggatcatcggactttgtcctgtggagtaagatcgcgaaacggtgaaagccgtaggtctctgctaaggtAAAGGAUCUaug

Caffeine 3 reverse >>>GCAT GGTCTC T CATAGATCCTTTACCTTAGCA >>>Melting temp: 52.9 °C >>>Self dimer ΔG: -5.4 kcal.mole-1 >>>Hairpin ΔG: -0.8 kcal.mole-1

Caffeine 4

aaatcataaaaaatttatttgctttgtgagcggataacaattataatagattcaattgtgagcggataacaattactagagatacgactcactataggtaccggatgtccagtcgcttgcaatgcccttttagaccctgatgaggatcatcggactttgtcctgtggagtaagatcgcgaaacggtgaaagccgtaggtctctgctaaggtAAtAtGCGGatg

Caffeine 4 reverse >>>GCAT GGTCTC A CATCCGCATATTACCTTAGC >>>Melting temp: 54.1 °C >>>Self dimer ΔG: -5.4 kcal.mole-1 >>>Hairpin ΔG: -0.2 kcal.mole-1

Caffeine 5 aaatcataaaaaatttatttgctttgtgagcggataacaattataatagattcaattgtgagcggataacaattactagagatacgactcactataggtaccggatgtccagtcgcttgcaatgcccttttagaccctgatgaggatcatcggactttgtcctgtggagtaagatcgcgaaacggtgaaagccgtaggtctctgctaaggtAAGGUGCCCaug

Caffeine 5 Reverse >>>GCAT GGTCTC G CATGGGCACCTTACCTTAGC >>>Melting temp: 58.2 °C >>>Self dimer ΔG: -5.4 kcal.mole-1 >>>Hairpin ΔG: -5.1 kcal.mole-1

Caffeine 6

aaatcataaaaaatttatttgctttgtgagcggataacaattataatagattcaattgtgagcggataacaattactagagatacgactcactataggtaccggatgtccagtcgcttgcaatgcccttttagaccctgatgaggatcatcggactttgtcctgtggagtaagatcgcgaaacggtgaaagccgtaggtctctgctaaggtAAGAtCCAGatg

Caffeine 6 Reverse >>>GCAT GGTCTC C CATCTGGATCTTACCTTAGC >>>Melting temp: 54.1 °C >>>Self dimer ΔG: -6.5 kcal.mole-1 >>>Hairpin ΔG: -1.6 kcal.mole-1

Caffeine 7 aaatcataaaaaatttatttgctttgtgagcggataacaattataatagattcaattgtgagcggataacaattactagagatacgactcactataggtaccggatgtccagtcgcttgcaatgcccttttagaccctgatgaggatcatcggactttgtcctgtggagtaagatcgcgaaacggtgaaagccgtaggtctctgctaaggtAACAGCUCGaug

Caffeine 7 Reverse >>>GCAT GGTCTC C CATCGAGCTGTTACCTTAGC >>>Melting temp: 56.2 °C >>>Self dimer ΔG: -6.8 kcal.mole-1 >>>Hairpin ΔG: -2.2 kcal.mole-1

Caffeine 8 aaatcataaaaaatttatttgctttgtgagcggataacaattataatagattcaattgtgagcggataacaattactagagatacgactcactataggtaccggatgtccagtcgcttgcaatgcccttttagaccctgatgaggatcatcggactttgtcctgtggagtaagatcgcgaaacggtgaaagccgtaggtctctgctaaggtAACAUACCGaug

Caffeine 8 Reverse >>>GCAT GGTCTC A CATCGGTATGTTACCTTAGC >>>Melting temp: 54.1 °C >>>Self dimer ΔG: -5.4 kcal.mole-1 >>>Hairpin ΔG: -2.3 kcal.mole-1

Caffeine 9 aaatcataaaaaatttatttgctttgtgagcggataacaattataatagattcaattgtgagcggataacaattactagagatacgactcactataggtaccggatgtccagtcgcttgcaatgcccttttagaccctgatgaggatcatcggactttgtcctgtggagtaagatcgcgaaacggtgaaagccgtaggtctctgctaaggtAAGGtGtAGatg

Caffeine 9 Reverse >>>GCAT GGTCTC T CATCTACACCTTACCTTAGC >>>Melting temp: 54.1 °C >>>Self dimer ΔG: -5.4 kcal.mole-1 >>>Hairpin ΔG: 0.0 kcal.mole-1

Caffeine 10 aaatcataaaaaatttatttgctttgtgagcggataacaattataatagattcaattgtgagcggataacaattactagagatacgactcactataggtaccggatgtccagtcgcttgcaatgcccttttagaccctgatgaggatcatcggactttgtcctgtggagtaagatcgcgaaacggtgaaagccgtaggtctctgctaaggtAAAGGtGtAatg

Caffeine 10 Reverse >>>GCAT GGTCTC T CATTACACCTTTACCTTAGCA >>>Melting temp: 52.9 °C >>>Self dimer ΔG: -5.4 kcal.mole-1 >>>Hairpin ΔG: 0.0 kcal.mole-1

Caffeine 11 aaatcataaaaaatttatttgctttgtgagcggataacaattataatagattcaattgtgagcggataacaattactagagatacgactcactataggtaccggatgtccagtcgcttgcaatgcccttttagaccctgatgaggatcatcggactttgtcctgtggagtaagatcgcgaaacggtgaaagccgtaggtctctgctaaggtAAtACGCCGatg

Caffeine 11 Reverse >>>GCAT GGTCTC G CATCGGCGTATTACCTTAGC >>>Melting temp: 56.2 °C >>>Self dimer ΔG: -6.8 kcal.mole-1 >>>Hairpin ΔG: -2.8 kcal.mole-1

Caffeine 12 aaatcataaaaaatttatttgctttgtgagcggataacaattataatagattcaattgtgagcggataacaattactagagatacgactcactataggtaccggatgtccagtcgcttgcaatgcccttttagaccctgatgaggatcatcggactttgtcctgtggagtaagatcgcgaaacggtgaaagccgtaggtctctgctaaggtAAtttACCGatg

Caffeine 12 Reverse >>>GCAT GGTCTC A CATCGGTAAATTACCTTAGCA >>>Melting temp: 52.9 °C >>>Self dimer ΔG: -7.3 kcal.mole-1 >>>Hairpin ΔG: -2.3 kcal.mole-1

Caffeine 13 aaatcataaaaaatttatttgctttgtgagcggataacaattataatagattcaattgtgagcggataacaattactagagatacgactcactataggtaccggatgtccagtcgcttgcaatgcccttttagaccctgatgaggatcatcggactttgtcctgtggagtaagatcgcgaaacggtgaaagccgtaggtctctgctaaggtAAAGtGtAGatg

Caffeine 13 Reverse >>>GCAT GGTCTC A CATCTACACTTTACCTTAGCA >>>Melting temp: 52.9 °C >>>Self dimer ΔG: -5.4 kcal.mole-1 >>>Hairpin ΔG: 0.0 kcal.mole-1

Caffeine 14 aaatcataaaaaatttatttgctttgtgagcggataacaattataatagattcaattgtgagcggataacaattactagagatacgactcactataggtaccggatgtccagtcgcttgcaatgcccttttagaccctgatgaggatcatcggactttgtcctgtggagtaagatcgcgaaacggtgaaagccgtaggtctctgctaaggtAAtGGtttGatg

Caffeine 14 Reverse >>>GCAT GGTCTC G CATCAAACCATTACCTTAGCA >>>Melting temp: 52.9 °C >>>Self dimer ΔG: -7.8 kcal.mole-1 >>>Hairpin ΔG: -2.0 kcal.mole-1

Caffeine 15 aaatcataaaaaatttatttgctttgtgagcggataacaattataatagattcaattgtgagcggataacaattactagagatacgactcactataggtaccggatgtccagtcgcttgcaatgcccttttagaccctgatgaggatcatcggactttgtcctgtggagtaagatcgcgaaacggtgaaagccgtaggtctctgctaaggtAAtAGtCCCatg

Caffeine 15 Reverse >>>GCAT GGTCTC C CATGGGACTATTACCTTAGC >>>Melting temp: 54.1 °C >>> Warning. This primer has a low ΔG value for self dimer. (-20.8 kcal.mole-1) >>>Hairpin ΔG: -3.9 kcal.mole-1

Caffeine 16 aaatcataaaaaatttatttgctttgtgagcggataacaattataatagattcaattgtgagcggataacaattactagagatacgactcactataggtaccggatgtccagtcgcttgcaatgcccttttagaccctgatgaggatcatcggactttgtcctgtggagtaagatcgcgaaacggtgaaagccgtaggtctctgctaaggtAAAGCCtAGatg

Caffeine 16 Reverse >>>GCAT GGTCTC A CATCTAGGCTTTACCTTAGC >>>Melting temp: 54.1 °C >>>Self dimer ΔG: -5.4 kcal.mole-1 >>>Hairpin ΔG: -1.1 kcal.mole-1

Caffeine 17 aaatcataaaaaatttatttgctttgtgagcggataacaattataatagattcaattgtgagcggataacaattactagagatacgactcactataggtaccggatgtccagtcgcttgcaatgcccttttagaccctgatgaggatcatcggactttgtcctgtggagtaagatcgcgaaacggtgaaagccgtaggtctctgctaaggtAAtAGtttGatg

Caffeine 17 Reverse >>>GCAT GGTCTC G CATCAAACTATTACCTTAGCAG >>>Melting temp: 53.6 °C >>>Self dimer ΔG: -5.4 kcal.mole-1 >>>Hairpin ΔG: 0.0 kcal.mole-1

We will ordered the primers today and then I can start working on the getting the plasmid ready by growing up J10079. Also, I will finish looking over MO West xanthine aptamers.

I started 6 3ml cultures of J100079 (Box 8-2 #100) in LB Amp. I grew the cultures o/n at 37C shaking. Tomorrow I will miniprepr each culture and save for iPCR when primers arrive.

cadoyle, 17 January 2014

My objective today is to miniprep cultures, finish examining the xanthine riboswitches, and continue to work on editing the thesis.

I performed a miniprep of each of the 6 cultures according to [29].

I combined all 6 minpreprs with a final volume of 118ul. I nanodropped the final volume and got: 47.9 ng/ul 260: 0.975 280: 0.545 260/280: 1.75 260/230: 1.52

I will save the miniprep for iPCR.

I ran the xanthine riboswitches through the basechecker.py and found none the RBS's or start codons of the riboswitches base paired to the aptamer. Also, earlier I ran the switches through test.py and the difference in free energy of each riboswitch between the ON and OFF state is greater the binding energy of the xanthine aptamer. I emailed Dr. Campbell to let him know.

cadoyle, 18 January 2014

Today I finished writing up paragraph summaries for all the biology seminars I attended last semester. Also, I worked on editing my thesis and adding the primers I designed this week to the methods section.

cadoyle, 19-20 January 2014

I worked on editing my thesis. I finished all the edits from the draft due at the end of last semester. Also, I added what did last week with the primers and riboswitch synthesis designs. The primers arrived today. I stored them the -20C freezer in the lab. I ran a PCR reaction on J19007 the receiving plasmid with primers J19007 G- Forward and J19007 reverse following this protocol [30]. The Tm for forward was 67 and for the reverse was 61. I set the annealing temp to 56 and elongation to 3:30min because the final product should 2,984bp. Tomorrow I will run a gel to verify iPCR size.

cadoyle, 21 January 2014

Gel to verify iPCR product: I made a 0.4% gel using 0.24g of agarose and 60ml of 0.5X TBE. I added 2ul of Midori green and cooled. I cleaned the gel box and added new 0.5X TBE buffer. In lane 1 I put 5ul of 1kb ladder. Lane 2: 5ul of J100079 Lane 3: 5ul of J10079 iPCR. I ran the gel at 100V for 30 minutes.

The control of J100079 is at about 3119bp (lane2) and the iPCR product is little smaller at about 2984bp (lane 3). Amplification and the size of the product looks good. I will clean up the PCR product and save for GGA ligation.

Ethanol Precipitation of iPCR product Following this protocol I cleaned the iPCR product for GGA ligation [31]. I let the product incubate -70C for 20 minutes and resuspended in 20ul of water.

Nanodrop of cleaned iPCR product: 1556.3 ng/ul 260: 31.126 280: 16.949 260/280: 1.84 260/230: 2.34

The values look great. Amplification worked well. I will save plasmid for GGA ligation once the G-block riboswitches arrive. My final volume is 18ul.

cadoyle, 22 January 2014

Riboswitches should arrive today. This is plan of action for the next tow weeks. Things are subject to change.

I have a question on the best way to test the constructs. I am thinking liquid but will talk to Dr. C today about it. I will need to check if we have enough plates and media etc.

We have enough plates for a least a couple of weeks.

The riboswitches arrived. I need to re-suspend them in TE to last up to 24 months. If I re-suspend in water they will only last for a month.

I made 10X TE stock: 5ml of 1M Tris, pH 8.0 1ml of 0.5M EDTA 44ml of water

I then diluted the stock to 1X TE I took 10mls of 10X and 90ml of water

To re-suspend the riboswitches I centrifuged the tube for 3−5 sec at a minimum of 3000 x g to pellet the material to the bottom of the tube. I added 20 μL TE to the tube for a final concentration of 10 ng/μL. Briefly vortex and centrifuged. GGA of Caffeine and Theophylline riboswitch with J100079 Plasmid

1 µL (50 ng) plasmid containing J100079 5µL dH2O 
1 µL 10X Promega Ligase Buffer
 1 µL 500 mM KOAc 
0.5 µL Bsa I high fidelity (HF) restriction enzyme 
0.5 µL T4 DNA Ligase 
9 µL final volume 


I added 1 ul of 10ng/ul of caffeine and theophylline riboswitches to separate tubes. I also did a control with just plasmid. I added 1ul of water in place of the riboswitch for the control. I put the tubes into the PCR machine. 
I program it for the following cylces: ▪ 20 cycles of 37C for 1 minute/16C for 1 minute ▪ 1 cycle of 37C for 15 minutes ▪ 22C holding temperature After the cycles are finished I will go straight to transformation.

Transformation of Riboswitches: 1) I thawed the zippy competent cells on ice for 5 minutes. Each tube contains 50 µL of cells. 2) I aliquot 50ul of cells into a cold test tube 3) I added 10µL of GGA ligation mixture 4) I incubated the ligation and cell mixture on ice for 5 minutes. 5) I Add 40ul of SOC media to a final volume of 100 µL. 6) I spread the cells onto LB Amp plates 7) I placed the plates in a 37C incubator overnight

Tomorrow I will perform a colony PCR to verify if the ligation and transformation were successful.

cadoyle, 23 January 2014

I check the transformations. The control had three small colonies. Caffeine and theophylline riboswitch both had one very small colony. Either it needs more time to grow or the plates are bad. I will let them grow a little longer. The plates were made in September and the antibiotic resistance might be gone. I can check the plates by just plating some cells on them and see if they grow today. I might need to make more plates.

I talked to Dr. Campbell and I might have had to much insert for the ratio to plasmid. I had about 3:1 ration of insert to plasmid. GGA was originally designed for 1:1. The insert is about 200bp and the plasmid is about 3000bp. 3000/200 is 16%. My plasmid is 50ng/ul so if divide 16/5 I get 3.2. I want a 1/1 so if I divide 10ng/ul be 3.2 I get that I need 3.2ng of insert to 50ng of plasmid to have a 1/1 ratio by weight.

It is also possible that the TE could be causing problem, since it contains EDTA.

Dilution of riboswitches to 3.2ng of insert: I took 1.6ul of both the caffeine and theophylline riboswitch and mixed with 3.4ul of water.

GGA of Caffeine and Theophylline riboswitch with J100079 Plasmid

1 µL (50 ng) plasmid containing J100079 5µL dH2O 
1 µL 10X Promega Ligase Buffer
 1 µL 500 mM KOAc 
0.5 µL Bsa I high fidelity (HF) restriction enzyme 
0.5 µL T4 DNA Ligase 
9 µL final volume 


cadoyle, 24 January 2014

I looked at the transformation from yesterday. The Theophylline riboswitch transformation has one colony. The caffeine riboswitch does not have any colonies. The control does not have any colonies either.

I am going to grow the colony from the theophylline riboswitch transformation. Tomorrow I will minprep and digest. Then run on a gel to verify the size. It will be hard to colony PCR because I don’t have a control to compare it to.

Saving theophylline riboswitch colony:

I placed 3mls of Lb Amp in a test tube. I picked the colony and inoculated it the in the media. I grew the culture overnight at 37C shaking.

I am not sure what is wrong with GGA for caffeine. I can re do the GGA ligation. I talked to Dr. Campbell and it is possible that the T4 ligase or ligase buffer is old. I am going to try another GGA ligation with new T4 ligase and buffer. It is also possible that the BsaI is bad. If the GGA ligation does not work the next time then I will have to wait until the BsaI arrives.

GGA of Caffeine riboswitch with J100079 Plasmid

1 µL (50 ng) plasmid containing J100079 5µL dH2O 
1 µL 10X Promega Ligase Buffer
 1 µL 500 mM KOAc 
0.5 µL Bsa I high fidelity (HF) restriction enzyme 
0.5 µL T4 DNA Ligase 
9 µL final volume 


I added 1 ul of 3.2ng/ul of caffeine riboswitches to separate tubes. I also did a control with just plasmid. I added 1ul of water in place of the riboswitch for the control. I put the tubes into the PCR machine. 
I program it for the following cylces: ▪ 30 cycles of 37C for 1 minute/16C for 1 minute ▪ 1 cycle of 37C for 15 minutes ▪ 22C holding temperature After the cycles are finished I will go straight to transformation.

Transformation of Riboswitches: 1) I thawed the zippy competent cells on ice for 5 minutes. Each tube contains 50 µL of cells. 2) I aliquot 50ul of cells into a cold test tube 3) I added 10µL of GGA ligation mixture 4) I incubated the ligation and cell mixture on ice for 5 minutes. 5) I Add 40ul of SOC media to a final volume of 100 µL. 6) I spread the cells onto LB Amp plates 7) I placed the plates in a 37C incubator overnight

Tomorrow I will perform a colony PCR to verify if the ligation and transformation were successful.

I added 1 ul of 3.2ng/ul of caffeine and theophylline riboswitches to separate tubes. I also did a control with just plasmid. I added 1ul of water in place of the riboswitch for the control. I put the tubes into the PCR machine. 
I program it for the following cylces: ▪ 20 cycles of 37C for 1 minute/16C for 1 minute ▪ 1 cycle of 37C for 15 minutes ▪ 22C holding temperature After the cycles are finished I will go straight to transformation.

Transformation of Riboswitches: 1) I thawed the zippy competent cells on ice for 5 minutes. Each tube contains 50 µL of cells. 2) I aliquot 50ul of cells into a cold test tube 3) I added 10µL of GGA ligation mixture 4) I incubated the ligation and cell mixture on ice for 5 minutes. 5) I Add 40ul of SOC media to a final volume of 100 µL. 6) I spread the cells onto LB Amp plates 7) I placed the plates in a 37C incubator overnight

Tomorrow I will perform a colony PCR to verify if the ligation and transformation were successful.