Generator

Part:BBa_K606018:Experience

Designed by: Cyrille Pauthenier   Group: iGEM11_Paris_Bettencourt   (2011-07-07)

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.


Microscopy Characterization

We put our T7 autoloop construct in in BL21 strains of E.coli, hosted in the plasmid pSB1C3.

BL21 strains are E.coli cells producing T7 RNA polymerase upon IPTG induction. You can also use glucose as an inhibitor of the system.

Here we used a simple protocol to make sure that we have nice positive and negative controls for furhter experiments. What follows shows that our cells are able to keep a memory of an active state.

Negative Control

First we launch cells from the overnight culture tube without IPGT. Then we wash the cells and relaunch them with glucose to inhibit out gene expression.

E.coli BL21 strains expressing the T7 polymerase at 37°C
E.coli BL21 strains with glucose (for inhibition of T7 RNA polymerase production) at 37°C -/- (trans image)
E.coli BL21 strains with glucose (for inhibition of T7 RNA polymerase production) at 37°C -/- (gfp image)

Here we can see the minimal leak of the GFP expression in BL21, in the right top of the picture we can see a cell where our T7 autoloop is strongly activated.

Positive Control

First we launch cells from the overnight culture tube. Cells are here induced with IPGT. Then we wash the cells and relaunch them with IPTG. This way we are sure that the T7 RNA polymerase gene of BL21 gene will be expressed.


E.coli BL21 strains expressing the T7 polymerase at 37°C
E.coli BL21 strains (IPTG induced for T7 polymerase expression) at 37°C +/+ (trans image)
E.coli BL21 strains (IPTG induced for T7 polymerase expression) at 37°C +/+ (gfp image)


Here we can see the activated cells. Their GFP expression is at the maximum the T7 autoloop allows.

Testing the memory of the autoloop

1-First we launch cells from the overnight culture tube without IPGT. Then we wash the cells and relaunch them with IPTG.

E.coli BL21 strains expressing the T7 polymerase at 37°C
E.coli BL21 strains (not grown with IPTG but IPTG induced when plated for T7 polymerase expression) at 37°C -/+ (trans image)
E.coli BL21 strains (not grown with IPTG but IPTG induced when plated for T7 polymerase expression) at 37°C -/+ (gfp image)

Our pictures where not very clear because of a focus issue with this specific slide. However, you can still see a somewhat strong fluorescence, indicating induction on the solid medium.


2-First we launch cells from the overnight culturetube. Cells are induced with IPGT. Then we wash the cells and relaunch them with glucose to inhibit T7 RNA polymerase expression in BL21.

E.coli BL21 strains expressing the T7 polymerase at 37°C
E.coli BL21 (grown with IPTG but glucose-inhbited when plated for T7 polymerase expression in the tube but nopt when plated) at 37°C +/- (trans image)
E.coli BL21 strains (grown with IPTG but glucose-inhbited when plated for T7 polymerase expression in the tube but nopt when plated) at 37°C +/- (gfp image)


Here cells are strongly glowing. That means they show some ability to keep their expression even after repressing the expression of T7 polymerase.

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