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Team:Paris Bettencourt/Experiments/T7 diffusion experiments
From 2011.igem.org
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<h2>Summary</h2> | <h2>Summary</h2> | ||
<h2>Emitter construct in <i>E.coli</i> - Receiver construct in <i>B.subtilis</i> (plasmid)</h2> | <h2>Emitter construct in <i>E.coli</i> - Receiver construct in <i>B.subtilis</i> (plasmid)</h2> | ||
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| + | As a control for Coli to Subtillis diffusion we wanted to use E.Coli as an T7 RNA Polymerase emitter and B.Subtillis as theT7 autoloop reciever. | ||
| + | Here we can see pictures taken using our classic microscopy protocol. | ||
| + | |||
| + | {| border="1" class="wikitable" style="text-align: center;" | ||
| + | |+T7 emitter RFP + Control in B.subtilis at 37°C | ||
| + | |- | ||
| + | |[[File:T7emColi T7autoBS trans 0.jpg|450px|thumb|center|Coli T7 emiter/ B.Subtillis T7 autoloop at 37°C (trans image)]] | ||
| + | |[[File:T7emColi T7autoBS fluo 0.jpg|450px|thumb|center|Coli T7 emiter/ B.Subtillis T7 autoloop at 37°C (gfp image)]] | ||
| + | |- | ||
| + | |[[File:T7emColi T7autoBS trans 125.jpg|450px|thumb|center|Coli T7 emiter/ B.Subtillis T7 autoloop after 125min at 37°C (trans image)]] | ||
| + | |[[File:T7emColi T7autoBS fluo 125.jpg|450px|thumb|center|Coli T7 emiter/ B.Subtillis T7 autoloop after 125min at 37°C (rfp image)]] | ||
| + | |} | ||
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| + | As the pictures show, there is no new highly glowing cell after 125 minutes experimentation. | ||
<h2>Emitter & receiver constructs in <i>B.subtilis</i> (receiver in plasmid)</h2> | <h2>Emitter & receiver constructs in <i>B.subtilis</i> (receiver in plasmid)</h2> | ||
Revision as of 00:19, 29 October 2011
Testing nanotubes with T7 RNA polymerase diffusion
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.
Summary
Emitter construct in E.coli - Receiver construct in B.subtilis (plasmid)
As a control for Coli to Subtillis diffusion we wanted to use E.Coli as an T7 RNA Polymerase emitter and B.Subtillis as theT7 autoloop reciever. Here we can see pictures taken using our classic microscopy protocol. {| border="1" class="wikitable" style="text-align: center;" |+T7 emitter RFP + Control in B.subtilis at 37°C |- |[[File:T7emColi T7autoBS trans 0.jpg|450px|thumb|center|Coli T7 emiter/ B.Subtillis T7 autoloop at 37°C (trans image)]] |[[File:T7emColi T7autoBS fluo 0.jpg|450px|thumb|center|Coli T7 emiter/ B.Subtillis T7 autoloop at 37°C (gfp image)]] |- |[[File:T7emColi T7autoBS trans 125.jpg|450px|thumb|center|Coli T7 emiter/ B.Subtillis T7 autoloop after 125min at 37°C (trans image)]] |[[File:T7emColi T7autoBS fluo 125.jpg|450px|thumb|center|Coli T7 emiter/ B.Subtillis T7 autoloop after 125min at 37°C (rfp image)]] |} As the pictures show, there is no new highly glowing cell after 125 minutes experimentation.Emitter & receiver constructs in B.subtilis (receiver in plasmid)
Emitter & receiver constructs in B.subtilis (receiver in genome)
We used our microscopy protocol to do this experiment. The emitter strain is a B.subtilis PY9 strain where our emitter construct is integrated in the genome. The receiver strain is a B.subtilis PY9 strain where our T7 autoloop (receiver) construct is integrated in the genome.
We followed our cultures on three microscopic slides: one for the emitter, one for the receiver and one for the mix.
Using the microfluidic chip (B.subtilis/B.subtilis)
Concentrating the cells more for microscopy (B.subtilis/B.subtilis)
Conclusions
Diffusion experiments
Results negative at first
This design was successfully implemented both in E.coli and B.subtilis. We were therefore able to test the diffusion of the T7 RNA polymerase through nanotubes. We ran several experiments:
- Diffusion from E.coli to B.subtilis with our receptor construct on a plasmid. Find more about it here
- Diffusion from B.subtilis to B.subtilis with our receptor construct on a plasmid. Find more about it here.
- Diffusion from B.subtilis to B.subtilis with our receptor construct integrated in the genome. Find more about it here.
The first three experiments gave us negative results. We saw no obvious increase of the GFP expression in receiver cells when our construct was on a plasmid and absolutely no GFP fluorescence when it was integrated in the genome.
Unexepected breakthrough
However, our microfluidic experiment gave unexepected and encouraging results. We used for this experiment two B.subtilis strains (one emitter, one receiver, both integrated in the genome). Our chromosomic T7 autoloop was brightly activated during this experiment, but only in densely packed mix of emitter and receiver cells. Find more about this experiment here.
Seeing that cell concentration seemed to be the key factor and taking advantage of our perfectly not leaky chromosomic autoloop we conducted a final set of experiments where we concentrated our cells even more. We invite you to see our final results here.
