Difference between revisions of "Will Notebook1"

Revision as of 18:35, 25 September 2008

Back to Main Page

Wideloache 13:59, 25 September 2008 (EDT)

Today I began the construction of the two Lux promoters - pLux and pLuxLas. These promoters will be constructed using primer extension. The primers used overlapped 20 bps on the 3' end. I used the following PCR protocol to extend the primers:

1 ug of primer 1
1 ug of primer 2
18 ul of H₂0
20 ul of Monster Mix

PCR Program = PrimerEx:

1. 5 mins at 94 C
   
2. 30 secs at 94 C
   
3. 15 secs at 55 C
   
4. 30 secs at 74 C
   
5. Repeat steps 2-4 for 4X
   
6. 5 mins at 74 C
   
7. Infinity at 21 C
   

I then did a Zymo cleanup of the PCR reaction and and eluted into 10 uL of H₂0. I then digested the two PCR extensions with EcoRI and PstI:

5 uL of DNA
11 uL of H₂0
2 uL of Buffer H
1 uL of EcoRI
1 uL of PstI

Wideloache 14:39, 23 September 2008 (EDT)

I ran the digestions on a .8% gel, and obtained the following result:

1=S03954(E/S), 2=B0015(E/X), 3=J23100(S/P), 4=S03983(X/P)

At this point I realized that I had a couple of problems:

Problem 1: At some point along the way, I switched the Registry #'s of S03954 (LasR-TT) and S03983 (Prom-LuxR) with their descriptions. Therefore, I had done the incorrect digestion for each of these parts. S03954 needs to be digested as a back insert (and then ligated with J23100) while S03983 needs to be digested as a front insert (and then ligated with B0015).

Problem 2: The J23100 part that I digested with S/P gave an unexpected band at ~1000bp. I went back and looked at the documentation for this part and found that in the registry, it exists in the plasmid J61002. This plasmid has RFP between the Spe and Pst sites. I need to determine if the part was moved before ligating it with LuxR and other parts. If so, I need to locate this sample or else move it again myself into pSB1A2. If not, then I may have to redo the assembly of S03983.

After talking with Dr. Campbell, it appeared likely that the J23100 was indeed in the incorrect plasmid (J61002). I decided to sequence the 4 different parts that I was assembling to confirm what I had. I sequenced S03954 and S03983 with VR. I also sequenced S03983, B0015, and J23100 with VF2. While I wait for the results of that sequencing i will go ahead with primer dimer assembly.

make freezer stocks of:
S03954
J23100
B0015
Ec100D::pir116 cells from Chris

Wideloache 13:58, 22 September 2008 (EDT)

Last night I grew up S03983 (LasR-TT) in LB. Today I did a Zyppy MP and obtained a concentration of 139.9 ng/uL. I then performed the following digestions:

13 uL H20
3 uL DNA
2 uL Buffer
1 uL RE 1
1 uL RE 2

S03954 -> Front Insert (E/S) + B0015 -> Front Vector (E/X)

J23100 -> Back Vector (S/P) + S03983 -> Back Insert (X/P)

This is the first of two rounds of ligation need to make the construct: Prom-LuxR-TT-Prom-LasR-TT. This will be inserted into the genome using the plasmids shipped from the Anderson Lab last week.

Wideloache 14:44, 19 September 2008 (EDT)

I obtained minipreps of B0015, J23100, and S03954 (Promoter-LuxR) from the iGEM racks. I couldn't find a miniprep of S03983 (LasR-TT), but I got a sample from the -80. Note: S03954 was not in the -80 as far as I could tell. I will freeze this down along with the other MP's after I transform them into cells.

I also received the shipment from Chris and my oligos were shipped today. I will begin with pLas assembly and LuxR/LasR genome insertions this weekend.

Wideloache 14:03, 18 September 2008 (EDT)

Over the past few days, I have been getting up to speed on the project as a whole. A lot of time was spent figuring out the primer sequences for the pLux' and pLuxLas promoters. I found that the primers that had been ordered over the summer used the -10 region from the registry, which differed from the standard consensus sequence in E. coli. I also found that a tube switch had occurred over the summer since the sequences for pLux' matched the expected sequence for pLuxLas and vice versa. Still, all 6 clones had at least one mutation in them. I decided to order new primers with the consensus -10 region. This only required that I reorder the reverse primers. I will reuse the forward primers that were designed over the summer.

I also contact Chris Anderson about performing a genomic insertion of LuxR and LasR. He is in the process of shipping the necessary plasmids to me. I will be doing a phi80 genome integration (protocol).

Wideloache 16:20, 10 September 2008 (EDT)

On Monday, I got K091136, K091146, and K091117 out of the -80. I need to make pLux' and pLux+Las- and do a genomic insertion of LuxR/LasR before I can test the XOR promoters.

Begin Primer Synthesis of pLux' and pLux+Las- (could consider site directed mutagensis with Pallavi's primers)
Email Chris about genomic insert vector
Talk to Erin Feeney about the status of the LuxR and LasR cassettes