Team:MSU-Michigan/Demonstrate

Demonstrate

MSU iGEM has succeeded in inducing current from induction of a selected chemical.

We measured the results using both IPTG and current.

Below is a video showing the working bioreactors outputting current.

IPTG Induction Results

Current from Induction

Current correlating to YouTube video

Current graph correlating to the YouTube video above. This current is also displayed under Measurement to compare current as a reporter for protein expression to GFP fluorescence under anaerobic conditions.

deltamtrB_GFPmtrB Induced V Uninduced

Same full vector strain but induction at point shown displaying the actuality of IPTG inducing current increase. This shows the difference between background expression and actual induction very distinctly.

deltamtrB_GFPmtrB Induced V Uninduced

The earlier graph compared to the one above. Shows how viability of cells earlier on produces a larger current increase compared to background expression.

Multiple Strains

Induction occurring at around 22 hours showing a doubling in current. The background control strains are shown here which display how little current is produced when mtrB is not present.

Contaminant Induction

Nitrate GFP Induction

Induction displayed very clearly by optimal concentration levels of nitrate. It appears that contaminant levels > .5µM prove to be toxic to the cells. IPTG was added at the beginning at 100µM concentration.

Paraquat GFP Induction

Induction displayed very clearly by optimal concentration levels of hydrogen peroxide. Hydrogen peroxide was used as an inducer instead of Paraquat for safety so the promoter appears to be sensitive to multiple reactive oxygen species. It appears that contaminant levels > .5µM prove to be toxic to the cells. IPTG was added at the beginning at 100µM concentration.

Copper GFP Induction

Induction displayed very clearly by optimal concentration levels of copper. With increasing concentration of copper added up to the toxic level determined to be .1µM, GFP fluorescence increases with increasing inducer concentration. IPTG was added at the beginning at 100µM concentration.

Molybdenum GFP Induction

Induction displayed very clearly by optimal concentration levels of molybdenum. It appears that induction might have occurred with molybdenum but it is unclear (See discussion). IPTG was added at the beginning at 100µM concentration

Discussion

After we obtained initial results with IPTG induction, we began inserting the contaminant promoters in place of the T7 promoter and the lac operator. We began testing induction of these promoters in 96 well plates because we could run more strains compared to testing current in bioreactors. Initially, we could not induce GFP expression with just the contaminants. We then tested to see if the strains were could still be induced by IPTG. This would tell us if our confirmed sequencing results for the contaminant promoter in the place of the T7 promoter and lac operator was actually correct. We found that we could induce the new strains with IPTG. Still not satisfied, we induced with IPTG then the contaminant by suggestion of Dr. Michaela TerAvest to test for a possible two stage inducible system ("And-Gate" system). We obtained GFP induction with this system of induction so we developed a step-wise overview to explain the success of our project. We unfortunately ran out of time and potentiostat space to test contaminant induction with current.

Step-wise Overview of lac operon and reasoning for "And-Gate" System

1. PlacIQ promoter is always on (constitutive promoter)
2. This causes transcription of LacI gene
3. This causes translation and therefore the lac repressor to be made
4. The repressor then binds to the lac operator which inhibits the RNA polymerase bound at the T7 promoter from transcribing a gene
5. IPTG binds to the repressor and removes it thus allowing the RNA polymerase to transcribe
6. We cut out the T7 promoter and the known lac operator thus allowing for insertion of our promoter to detect contaminants
7. We accomplished that successfully with sequencing confirming that only the contaminant promoter is currently there
8. Yet, we see inducible current production and GFP expression by IPTG with these new strains
9. Therefore, a cryptic operator must exist on the plasmid that causes regulation of our contaminant promoter
10. We must add IPTG to remove the repressor before adding the contaminant thus displaying a possible two-stage inducible system ("And-Gate") )(1,2,3)
11. Typically, these systems are designed intentionally but ours appears to be incidental due to the cryptic operator (1,2,3)

Anaerobic Current Leading to Idea for Measurement

See button below the Graph

Anaerobic Current

Anaerobic current production correlating to the video above. These triplicate runs led the team to consider the idea of using current as a replacement for GFP as a reporter of gene expression under anaerobic conditions.

Measurement

References

(1) Khalil, A. S.; Collins, J. J. Nat. Rev. Genet. 2010, 11 (5), 367–379.
(2) Moon, T. S.; Lou, C.; Tamsir, A.; Stanton, B. C.; Voigt, C. A. Nature 2012, 491 (7423), 249–253.
(3) Purnick, P. E. M.; Weiss, R. Nat. Rev. Mol. Cell Biol. 2009, 10 (6), 410–422.

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