Difference between revisions of "IGEM 2009 notebook"
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I started out the day looking at my time-sensitive group research qustion about reporter proteins. I started making a table listing the advantages and disadvantages of different reporter proteins. Then the group found out that there was a major roadblock to our project: a stop codon was located in our ATG-5mer oligo. The stop codon would have been part of the BioBrick scar. The group looked at several ways to work around this problem. The idea that I looked most into was replacing the restriction sites that were used in the scar through standard assembly. According to the judging criteria for iGEM, I found that a team could possibly alter the standard assembly method as long as they wrote a letter to the iGEM judges explaining our plan in a detailed manner. From there, I set off to understand the BioBrick scar more by making a document highlighting the exact positions of the rsetriction sites on the BioBrick parts and how they were used to put more than one part together. I then found several other pairs of restriction sites that complemented one another and could be used in place of the standard scar between Xba1 and Spe1. At the end of the day, the group got the chance to speak with Dr. Campbell and he suggested a hybrid idea which would combine Davidson's restriction site idea with Missouri's PCR idea. I'm not quite sure of what this idea is exactly yet, but we'll all clarify our vague idea of it tomorrow morning after a talk with Dr. Eckdahl. | I started out the day looking at my time-sensitive group research qustion about reporter proteins. I started making a table listing the advantages and disadvantages of different reporter proteins. Then the group found out that there was a major roadblock to our project: a stop codon was located in our ATG-5mer oligo. The stop codon would have been part of the BioBrick scar. The group looked at several ways to work around this problem. The idea that I looked most into was replacing the restriction sites that were used in the scar through standard assembly. According to the judging criteria for iGEM, I found that a team could possibly alter the standard assembly method as long as they wrote a letter to the iGEM judges explaining our plan in a detailed manner. From there, I set off to understand the BioBrick scar more by making a document highlighting the exact positions of the rsetriction sites on the BioBrick parts and how they were used to put more than one part together. I then found several other pairs of restriction sites that complemented one another and could be used in place of the standard scar between Xba1 and Spe1. At the end of the day, the group got the chance to speak with Dr. Campbell and he suggested a hybrid idea which would combine Davidson's restriction site idea with Missouri's PCR idea. I'm not quite sure of what this idea is exactly yet, but we'll all clarify our vague idea of it tomorrow morning after a talk with Dr. Eckdahl. | ||
Revision as of 14:40, 5 June 2009
Roclemente 09:57, 3 June 2009 (EDT)'
Shamita and I are trying to find suitable reporter proteins to use. Yesterday, Leland and Alyndria were working on ways to insert the gene sequences into the plasmid. Upon seeing how they wanted to manipulated the reporter gene to include the logical clauses, we came up with a few criteria for the reporter genes we would use. The following criteria for genes are listed in order of the broadest aspect to look at to the narrowest aspect:
a) Doesn't contain restriction sites for the 4 restriction enzymes (EcoR1, Xbal, Spel, Pst1) used to cleave the Biobrick part out of the plasmid.
b) Contains 6 cutter restriction sites.
c) These restriction enzymes aren't blunt (cleave straight down at one spot).
d) These restriction sites are close to thge 5' (beginning) end of the sequence.
e) These enzymes are easiest to work with and cheapest.
We are finding the part numbers of the reporter genes we want to use (antibiotic resistance, fluorescence, LacZ) through our own GCAT because we know these ones work. We are then locating these parts on the parts registry [1] website. We copied and pasted the gene sequences we obtained from the registry onto the ApE software [2]. From here, we were able to generate a genetic map of each gene that outlined each restriction site that fit our criteria. We put each genetic map, alongside the part number used, into a Word document:
http://gcat.davidson.edu/GcatWiki/images/0/0e/Restriction_Site_Mapping_on_Reporter_Genes.doc
Roclemente 16:54, 3 June 2009 (EDT)
So it looks like we've changed directions. The team is looking towards the wet lab portion of our research more now. Instead of simply speculating about which reporter proteins we think will work best and which tRNA suppressors to use, we're going to physically test it out ourselves. A few tasks at hand in the second part of this afternoon:
1. What are the DNA gene sequences of the 12 different tRNA suppressors we want to use?
2. What is the sequence of the DNA that is cleaved off of both ends of the tRNA when it is transcribed?
We e-mailed Anderson to find out the exact tRNA gene sequences because we had a hard time finding this information everywhere else. Most papers we found describing the 5-base codon tRNA suppressors simply referred to Anderson's paper [3]. Anderson replied near the end of the day with the gene sequence. According to his email, the only difference between different tRNA genes is the anticodon loop. Therefore, if we just substitute all the anticodon loops in the invariable part of the sequence, we have our different tRNA sequences. We plan on using the Lancilator to help us break these tRNA sequences into smaller oligonucleotides that can anneal at around the same temperature because it is not possible for us to order oligos that are greater than 70 nucleotides long.
Roclemente 17:21, 4 June 2009 (EDT)
I started out the day looking at my time-sensitive group research qustion about reporter proteins. I started making a table listing the advantages and disadvantages of different reporter proteins. Then the group found out that there was a major roadblock to our project: a stop codon was located in our ATG-5mer oligo. The stop codon would have been part of the BioBrick scar. The group looked at several ways to work around this problem. The idea that I looked most into was replacing the restriction sites that were used in the scar through standard assembly. According to the judging criteria for iGEM, I found that a team could possibly alter the standard assembly method as long as they wrote a letter to the iGEM judges explaining our plan in a detailed manner. From there, I set off to understand the BioBrick scar more by making a document highlighting the exact positions of the rsetriction sites on the BioBrick parts and how they were used to put more than one part together. I then found several other pairs of restriction sites that complemented one another and could be used in place of the standard scar between Xba1 and Spe1. At the end of the day, the group got the chance to speak with Dr. Campbell and he suggested a hybrid idea which would combine Davidson's restriction site idea with Missouri's PCR idea. I'm not quite sure of what this idea is exactly yet, but we'll all clarify our vague idea of it tomorrow morning after a talk with Dr. Eckdahl.
