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Experiments - T7 RNA polymerase diffusion

The T7 RNA polymerase diffusion design was the first one we thought of. It uses the RNA polymerase of the T7 phage, giving us perfect orthogonality to B.subtilis genome. We use an auto-amplification device base on this polymerase that we call T7 autoloop. This autoloop is also used in the tRNA amber diffusion design. We successfully BioBricked and characterized all our constructs for this design and made them available for the synthetic biology community. Our results are detailed below.

Abstract

Design overview

Schematic summary of the T7 diffusion device

All of the parts of the above design have been BioBricked, characterized both in E.coli and B.subtilis, and sent to the registry.

More details on the design are available here.

Parts and BioBricks construction

You can find the cloning plan for the T7 RNA polymerase design below:

INSERT CLONING PLAN

Setback with the pHyperspank promoter

At the beginning of the project, we intended to use a pHyperSpank promoter (Ph-s, BBa_K143055) in front of the emitter construct. The Ph-s promoter is repressed by LacI and would have allowed us to use an IPTG-inducible promoter. However, our first experiments with the promoter were inconclusive. We sequenced our sample and realized that we had received a BioBrick of the size of Ph-s but not the one we expected. It was anoter gene entirely. Rather than losing weeks of experiments waiting for another HyperSpank promoter, we chose to use a constitutive Pveg promoter (BBa_K143053).

Characterization of the T7 promoter

In order to characterize the pT7 promoter, we used the construct pT7-RBS-GFP-T7ter. This construct was transformed into BL21 strains (E.coli) expressing the T7 polymerase under IPTG induction.

Fluorescence kinetics

The measurements have been carried out on a spectrophotometer at 37°C under transient shaking. The experiment lasted 4h, we tested several colonies and several IPTG concentrations. The OD at 600nm and the fluorescence of the GFP (exc: 470nm / meas:515 nm) was measured every 5 min and the ratio of the two was calculated.

All values were normalized by substracting the fluorescence/OD value of the well with 0 mM IPTG at time 0. The values given are in arbitrary units.

After 2 hrs of induction, we see a clear increase of the fluorescence proportional to the IPTG concentration (that is to say with the quantity of T7 polymerase induced in the cell). After 4 hrs, the expression of GFP under the pT7 is still not saturated

Here we plot the ratio of induction of the T7 polymerase dependant construct for the different concentrations of IPTG at a given time (4 hrs) taking the well with 0 IPTG at time 0 as the reference.

Characterization of the T7 RFP emitter <--TO BE CORRECTED HEAVILY

We found out there is a small leakage with the T7 emitter RFP. The cells in which RFP production is activated stop dividing and glow very strongly.

T7 emitter RFP + Control in E.coli at 37°C

E.coli at 37°C (trans image)	E.coli at 37°C (rfp image)
E.coli negative control at 37°C (trans image)	E.coli negative control at 37°C (rfp image)

The first pictures show that the RFP construct is working efficiently since some cells are glowing with RFP fluorescence. This also shows the system is not as leaky as we expected. Indeed, the promoter regulating RFP expression is 'pVeg' which is a constitutive promoter. Finally, the RFP system is working very well because when leak occurs, cells glow very strongly.

Characterization of the T7 signal amplification leakage

We characterized T7 autoloop (receiver part of the construct, BBa_K606036) in E.coli, when hosted in the plasmid pSB1C3.

We know that, due to stochastic leakage, some cells should express T7 RNA polymerase even without induction. Our modeling suggests that only a few polymerases are required to activate the T7 autoloop. Without any induction, we therefore expected to have a few very bight cells (autoloop activated) while the other remain dark or only marginally fluorescent (bit of leakage on the GFP gene only).

We tried two configurations: one with a terminator before the pT7 promoter and one without. This was to see if we could reduce leakage with one extra terminator.

T7 autoloop in E.coli at 37°C

E.coli T7 autoloop without terminator at 37°C (trans image)	E.coli T7 autoloop without terminator at 37°C (gfp image)
E.coli T7 autoloop with terminator at 37°C (trans image)	E.coli T7 autoloop with terminator at 37°C (gfp image)
E.coli negative control at 37°C (trans image)	E.coli negative control at 37°C (gfp image)

These pictures show that the T7 GFP autoloop system is efficient since some cells are glowing with GFP fluorescence. Thus, we can conclude that the T7 autoloop is activated because of stochastic leakage.

Finally, we notice there is no difference between the GFP autoloop with and without terminator before the T7 promoter. It could be due to that terminator which is a B.subtilis terminator. Moreover, we know that this E.coli plasmid has 4 terminators before our construct, pretty much nullifying the effect of our extra terminator in E.coli.

Diffusion experiments