Difference between revisions of "Part:BBa K1913007"

Revision as of 11:23, 19 October 2016

434- and lambda cI operon for tuning protein balance

This part is intended to provide a balance between the 434- and lambda cI proteins depending on the strength of the RBS that is inserted upstream of 434 cI.

Usage and Biology

Before using this part, read the design notes. This part contains two viral genes: 434- and lambda cI (BBa_C0052 and part of BBa_K081007) expressed under the pBAD/AraC promoter (BBa_I0500). The 434 cI gene is not yet preceded by an RBS, allowing tuning of the cI protein balance. The 434 cI repressor protein encoded in this part should be more rapidly degraded (see design considerations) than the lambda cI protein. Therefore, changing the strength of the pBAD promoter in this part (with L-Arabinose or D-Glucose) should temporary shift the ratio between the two cI protein levels. We recommend using this part together with the modified lambda Prm promoter (BBa_I12006). Combining these parts should lead to interesting dynamics of expression under this modified lambda Prm promoter in response to changes in pBAD promoter strength.

We have combined BBa_K1913007 and BBa_K1913016 into BBa_K1913006, inserted an RBS library (see design notes) upstream of the 434 cI gene and transformed it to DH5alpha. The transformants were expected to produce subpopulations in mRFP expression upon glucose addition. We tested this with flowcytometry (Figure 1).

Figure 1. Flow cytometry results for clone B6 and the constitutive mRFP expression control. The images on the left are histograms that show counts of bacteria with specific values of forward light scatter (FSC-H). The histograms on the right show counts of cells with fluorescence corresponding to mRFP. The mRFP fluorescence was measured with an excitation at 561nm, measuring emission at 620nm with 10nm bandwidth.

Although flow cytometry shows that two clones do not form subpopulations, this is no reason to assume that none of the clones are able to do so. We chose to include an 18-mer RBS library on purpose. We expect that if the system described here can lead to subpopulations, this ability relies on the RBS strength. As we have not been able to measure all RBS inserts, we cannot accept nor reject the possibility that the system described here works as intended.

Sequence and Features