So far, trans activation has been tested for cis3 and cis4 (with their trans combinations 1 and 2), as well as trans3 with cis8, making the total count:
cis3trans1
cis3trans2
cis4trans1
cis4trans2
cis8trans3
The repressions and activation levels of viral protein was tested by replacing the viral protein gene with the reported molecule YFP, with fluorescence now an indicator of host cell protein concentration. Cells were prepared according to the flow cytometry protocol, and YFP fluorescence was measured with Quanta, with results below.
The fluorescence levels of D1210, YFP (the YFP construct with no cis repressor), and YFP + aTc serve as a control (as in the case of D1210, to show the fluorescence levels of background cells), as a guideline for minimum activation needed (as titering results show that N and Q levels in the (no-cis) construct are sufficient to cause lysis), and as maximum protein production possible (100% level set by the YFP + aTc results). Thus, the YFP fluorescence levels provide a estimate of target trans activation levels. Arabinose is added to the trans samples to induce trans production, and aTc added to increase production of the cis transcript.
cis3 and accompanying trans
Geometric mean was found for each cell type using FloJo:
cis3trans2 + ARAB + aTc : 163.67
cis3trans2 + ARAB : 11.63
cis3trans1 + ARAB + aTc : 159.38
cis3trans1 + ARAB : 12.05
cis3 + aTc : 32.75
cis3 : 4.05
YFP + aTc : 1308.11
YFP : 345.19
D1210 : 9.78
When activated by both arabinose and aTc, the geometric mean shows that cis3trans1 and cis3trans2 fluorescence levels reach almost half of the tetR repressed (no aTc) system. When compared to the 100% fluorescence value (set by the mean of the YFP + aTc culture), trans activation for these two combinations reach more than 10%, while cis repression (cis3 + aTc) is approximately 2%.
cis4 and accompanying trans
Geometric mean was found for each cell type using FloJo:
cis4trans2 + ARAB + aTc : 55.66
cis4trans2 + ARAB : 5.08
cis4trans1 + ARAB + aTc : 40.43
cis4trans1 + ARAB : 10.25
cis4 + aTc : 30.64
cis4 : 4.33
YFP + aTc : 1358.17
YFP : 327.47
D1210 : 4.89
Again, cis repression is relatively tight (cis4 repression even with aTc is less than 2% of maximum fluorescence, and without aTc is indistinguishable from background). Trans activation is not as high, however, being slightly less than one-sixth the value of YFP no-aTc, or about 3 to 4% activation, and not a significant increase over the 2% fluorescence level of the cis4 repressor.
cis8 and accompanying trans
Geometric mean was found for each cell type using FloJo:
cis8trans3 + ARAB + aTc : 49.63
cis8trans3 + ARAB : 4.02
cis8 + aTc : 32.38
cis8 : 4.04
YFP + aTc : 1358.86
YFP : 327.47
D1210 : 4.89
As with the other cis, cis8 repression is tight (2% with aTc). Trans activation levels are similar to the cis4 combinations, too, with cis8trans3 acting at about 3.5%.
Summary
The most promising results are for cis3 and its trans combinations, or cis3trans1 and cis3trans2, which show significant trans activation. The next step involves transforming the trans1 and trans2 plasmids into the cis3-containing Q construct, and titering. Hopefully, trans activation levels are enough to give plaques for the cis3trans1 and cis3trains2, thus showing that Q levels are high enough for successful phage infection.
