From 2007.igem.org

Western Meeting Notes

5/14 Overview of goals for the next two weeks

Begin Experimentation

Educational Information

Literature Research

5/15

Reviewed PowerPoint presentations of 2006 project to gain an understanding of the Pancake Problem.

Viewed Bruce's presentation on the mutation of the hix sites

Developed plan to build testing constructs of mutant hix sites, arriving this afternoon from Davidson by FedEx

Will build pLac - hix(n) - BBa_S03644 (RFPrev-RBSrev) - hix(n) on AmpR plasmid. Details to follow.

Note -- The above plan has changed as of Tuesday afternoon because of availability of parts.

BBa_S03644 is not available in our lab and the exact forward equivalent appears (amazingly) to not exist anywhere. However, there is an RBS - RFP in the Registry (BBa_I13502) which is a preassembled combination of a weaker RBS (BBa_B0034) and RFP (BBa_E1010). There is also a full positive control assembly (BBa_J04450) of pLac - B0034 - E1010 - Term. We will need to be careful to use the same parts in our Hix(n) assemblies so that we can test for only the functionality of our construct with the mutated Hix sites included.

5/16

We have cultures that were grown overnight of the Hix(n). We received the sequencing data on the Karen's and Bruce's Hix(n) constructs (from 5/14 report) and are analyzing the data to find the mutations. Lab work continues.

5/17

Karen developed a modified Green Fuorescent protein, GFP, by splitting the DNA for GFP, reversing one portion and adding Hix sites to it. This way, GFP can only be produced when the reversed portion is inverted so that the entire gene is in the correct order. This method was attempted with the TET resistance gene with no results. We have looked at using several fluorescent proteins modified in this manner to study the Traveling Salesman. In this manner, the proteins would only be expressed if they had all been lined up in the correct order. This approach has some flaws however, all fluorescent proteins are similar in structure, so that even if the portions of two different genes come together, the resulting protein may still fluoresce. Additionally, fluorescent protiens have the ability to undergo fluorescent resonant energy transfer, FRET, a phenomenon in which energy absorbed by one molecule is directly transfered to a second molecule, this prevents the first molecule from emitting visible photons, so that it may remain undetected.