Caltech/Project/Riboregulator Results

Quanta Data and Analyses

Cis repression has worked on every construct tested so far, reducing expression to nearly background levels. 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:

• Cis3/Trans1
• Cis3/Trans2
• Cis4/Trans1
• Cis4/Trans2
• Cis8/Trans3

Riboregulation was tested using YFP under the control of the tet-inducible system described here. The appropriate cis repressors were cloned in, replacing the spacer region. Those cells were cotransformed with plasmids containing the trans-activating RNA. Cells were prepared according to the flow cytometry protocol, and YFP fluorescence was measured by flow cytometry, as shown below. Arabinose is added to the trans samples to induce trans production, and aTc added to increase production of the cis transcript.

The fluorescence levels of D1210 (the negative control), the unrepressed/uninduced YFP construct, and YFP construct + aTc serve as a controls. Titering experience has shown that unrepressed/uninduced levels are sufficient for lysis. Therefore, the YFP fluorescence levels provide a estimate of target trans activation levels.

Fluorescence distributions of cis3-repressed, trans1-activated YFP. The x-axis shows fluorescent intensity in arbitrary units.

Geometric mean fluorescences were calculated using FlowJo software and are shown below. The background autofluorescence was subtracted from each value, except for the cis3-repressed, uninduced sample (in which the fluorescence was below background). Each construct was compared to the induced, unrepressed YFP fluorescence. Trans1 and trans2 both showed ~12-fold activation, from 1% to 12% of unrepressed expression.

Geometric mean fluorescence of cis3-repressed constructs, expressed as a fraction of the induced, unrepressed YFP mean fluorescence.

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%.