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

Revision as of 12:54, 9 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 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. This method is similar to the 2nd process described in the Inserting 5mers document. The only difference is the method in which the front segments are generated is PCR. 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
   RBS – 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
     ***Note 6/8/09 this TetR part was incorrect. We needed Tet resistance not the Tet repressor gene.

 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

--Leland 12:35, 8 June 2009 (EDT)

We finalized and ordered the primers and the tRNA suppressor gene oligos needed for our fist set of experiments Davidson Primers and Oligos. We then did a mini prep to prepare the bioparts we need (listed on previous post). The results for the ones I prepared are as shown.

 K091111 - 1 part solution/ 3 part dH2O
    Absorption @ 260 - 0.843
    260:280 - 2.00
    ng/µl - 42.1

 S035710 - 1 part solution/ 3 part dH2O
    Absorption @ 260 - 0.817
    260:280 - 1.98
    ng/µl - 40.9

Tomorrow Todo List
1. Finish with the cells in the tube
2. Finalize and upload the document on which tRNA suppressor we should use

--Leland 08:54, 9 June 2009 (EDT)

Tomorrow Todo List
1. Finish with the cells in the tube
2. Finalize and upload the document on which tRNA suppressor we should use