From 2007.igem.org

Now that we have an idea of which genes are “splittable” and therefore a number of nodes that we are able to work with, we can begin to build our first bacterial computer. There are three control experiments that our group has decided to conduct in conjunction with the construction of the bacterial computer.

The first experiment is to control that we will be able to see GFP and RFP at the same time in bacterial colonies

Plac:RBS:RFP:RBS:GFP

The second experiment is: if the problem is solved, will the construct show the desired phenotype.

T7:RBS:RFP1:hix:RFP2:RBS:GFP1:hix:GFP2

A separate plasmid containing the gene for the T7 RNA polymerase will be co-transformed with the constructs that use the T7 RNA polymerase promoter. Plac:RBS:T7 RNAP gene

The third deals with making sure that a polypeptide synthesized with different halves of the two different genes (becuase they are both fluorescent proteins) does not produce a postive phenotype.

Plac:RBS:GFP1:hix:RFP2 and Plac:RBS:RFP1:hix:GFP2

In conjunction with these controls we will synthesize our first bacterial computer

T7 RFP1-hix-[GFP2-RFP1]-hix-[GFP2-TT]-hix-[RFP2-GFP1]-hix

Note: The first half of each gene is ligated to an RBS

The orientation of nodes was decided upon by results from split-gene strength. Split RFP’s fluorescents is substantially less noticeable than GFP’s. We decided to ligate GFP after RFP in order to have a better chance of recognizing successfully split RFP’s phenotype. The orientation of the bacterial computer was decided upon by avoidance of “solved” phenotypes before flipping began.

Note: We will lastly be testing the T7 promoter, and the UV5 hybrid pLac promoter. The second control experiment will also function as a second promoter tester and we plan on comparing results from the three promoter testers (Davidson’s PT, MW PT, and the newest PT) to discern which method of controlling gene expression we will use. The ability to use Cre protein as a node is still in testing as well.