Generator
T7 auto-am

Part:BBa_K606036:Experience

Designed by: Axel Sguret   Group: iGEM11_Paris_Bettencourt   (2011-09-18)
Revision as of 13:48, 24 October 2011 by Hovannes (Talk | contribs) (→‎Negative Control)

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

Here we used a simple protocol to make sure that we have nice positive and negative controls. Glucose is used as an inhibitor of our system. Plus, what follows shows that our cells are able to memorise their activated state.

Negative Control

First we launch cells from the overnight's 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 expressing the T7 polymerase at 37°C -/- (trans image)
E.coli BL21 strains expressing the T7 polymerase 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 of which T7 way is strongly leaking.

Positive Control

irst we launch cells from the overnight's tube. Cells are induced with IPGT. Then we wash the cells and relaunch them with IPTG. This way we are sure that the gene will be expressed.


E.coli BL21 strains expressing the T7 polymerase at 37°C
E.coli BL21 strains expressing the T7 polymerase at 37°C -/- (trans image)
E.coli BL21 strains expressing the T7 polymerase at 37°C -/- (gfp image)

Mixed strategies

1-First we launch cells from the overnight's 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 expressing the T7 polymerase at 37°C -/- (trans image)
E.coli BL21 strains expressing the T7 polymerase at 37°C -/- (gfp image)

2-First we launch cells from the overnight's tube. Cells are induced with IPGT. Then we wash the cells and relaunch them with glucose.


E.coli BL21 strains expressing the T7 polymerase at 37°C
E.coli BL21 strains expressing the T7 polymerase at 37°C -/- (trans image)
E.coli BL21 strains expressing the T7 polymerase at 37°C -/- (gfp image)

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