Difference between revisions of "IGEM Notebook (6/2/09 - 7/31/09)"

Revision as of 15:01, 8 June 2009

--Leland 09:28, 2 June 2009 (EDT)

Today Alyndria and I worked on answering the question of how do we insert and construct the logical clauses (LCs) into the reporter gene. We outlined two processes for doing the LC inserts. One as a BioBrick, and the other only changing the first part of the gene. We feel the second one is much better because there are only two parts (of the gene) to be linked together, and only the first part has to be edited.

Tomorrow Todo List
1. Finalize and upload the document created today
2. Begin work on which tRNA suppressor we should use.

--Leland 17:35, 3 June 2009 (EDT)

I worked on putting together a document showing which tRNAs we should use. Will came up with an alternative method for how we will insert the logical clauses (or anything); however, we have yet to create a document showing this method. We decided we should order all the tRNAs except for the wobble one (11 total), so that we can begin testing them. I put together the ATG and 5mer sequences for preliminary tests on our system. These sequences will be used to test to see if our idea of having one suppressor tRNA bind to mRNA and alter the reading frame, allowing a protein to be expressed, will work. Davidson ATG+5mer BioBrick Sequences

Tomorrow Todo List
1. Finalize and upload the document on which tRNA suppressor we should use
2. Check the ATG+5mer sequences with the reporter genes with Shashank
3. Design the other surpressor tRNA encoding biobrick
4. Update the document created yesterday with the new plan

--Leland 10:13, 4 June 2009 (EDT)

Using Olivia's program, Shashank and I went through the genes RFP and Tet resistance looking for other 5mer sites (in the reading frame that would occur if no 5mer binds at the designed "LC" site). Thankfully, no unwanted 5mers show up in that reading frame. However, we discovered that in the scar in the reading frame if the 5mer does bind at the correct place, is TAG which is a stop codon (UAG). We spent the rest of the day working on this problem. I also updated tRNA doc. Inserting 5mers

Tomorrow Todo List
1. Choose a reporter gene... needs to have a restriction site early on
2. Work out ideas to overcome TAG (UAG) in reading frame
3. Finish Question
4. Finalize and upload the document on which tRNA suppressor we should use

--Leland 12:02, 5 June 2009 (EDT)

I fleshed out CR/Restriction enzyme solution to the TAG codon. Then Romina and I obtained the registry numbers for the cells we will need to begin culturing Sunday afternoon for Monday in the lab.

Promoter pLacIQ1 - K091112 968: 4-4, 49 969: 4-4, 50

pLac1Q - K091111 477: 3-4, 22 813: 1-4, 28

pLac + RBS – S03511 440: 3-2, 7

RBS RBS (strong) – B0030KH 874: 4-4, 14

RB – B0034 421: 2-1, 1 422: 2-1, 449: 2-1, 13 533: 1-3, 97 1056: 2-5, 57

Reporter Gene RFP - E1010 441: 2-1, 8 445: 2-1, 10

TetR – C0040 505: 3-4, 44 512: 3-4, 51

Optional things we may want pLsrR - K091114 812: 1-4, 27

pLac+RBS+RFP – I715039 426: 3-1, 1 429: 3-1, 2

Tomorrow Todo List
1. Design Primers
2. Do miniprep
3. Finalize and upload the document on which tRNA suppressor we should use