Virginia Tech/plasmid design

The right promoter of the λ switch, which we used to build our plasmid. We replaced the cI and Cro genes with fluorescent protein genes on our plasmid.

For our models, we needed to know how the virus infects a cell prior to seeing symptoms.

We decided to utilize the proteins that the virus naturally makes upon infection: cI and Cro. The decision between lysogeny and lysis is determined by a number of other proteins and conditions, but these are the final two proteins involved in the decision. Thus, for our reporter plasmid, we decided to use one of the promoters from bacteriophage lambda. This bistable promoter makes a decision based on the presence of these two proteins. However, on the reporter plasmid, instead of promoting the lytic or lysogenic pathway, the promoter will promote a different fluorescent gene depending on which protein is predominating.

Challenges in Design The lambda switch has two main regions: a left side and a right one. Each region contains two promoters that promote in opposite directions. We know that cI and Cro bind to the operators of the right region but they will also form a complex with the left one. We needed to use the promoters on the right region, but we were not sure to what extent binding on the left would affect our promoters. Thus, we decided to leave off the left region assuming that the promoters on the right will work just fine without it.

One of the two promoters in our right region, Prm, did not have a ribosome binding site. Therefore, translation started right after the +1 site. The other promoter, Pr, did have a ribosome binding site. To account for our uncertainty about RBS, we designed a multiple different promoters.

Construct 1: pSBVT0001

• iGEM prefix (EcoRI, NotI, XbaI) and suffix (SpeI, NotI, PstI)
• Original RBS from Pr, which we also copied over to the Prm in case an RBS was necessary to express the fluorescent proteins.
• Translational stop codon six base pairs after the +1 site on the Prm side so that translation will not start at the +1 like it would in the lambda genome naturally.

Construct 2: pSBVT0002

• iGEM prefix (EcoRI, NotI, XbaI) and suffix (SpeI, NotI, PstI)
• No extra RBS.iGEM provides ribosome binding sites as a part of its DNA registry and most of the reporter genes already have RBS built into the parts.
• Translational stop codon six base pairs after the +1 site on the Prm side.