Difference between revisions of "Part:BBa K415301"

Latest revision as of 03:44, 26 October 2010

Low Power Toggle - Ptrc:cI:tt:Plambda:LacI:tt

The original pTSMa plasmid from the Collins paper. This plasmid has been modified so that it now contains cI that is hypersensitive to cleavage by RecA = Low Power Toggle.

From the [http://2010.igem.org/Team:MIT 2010 MIT iGEM Team].

This part is a variation of a [http://www.pnas.org/content/101/22/8414.full Collins/Kobayashi UV Toggle] (2004) from an article in PNAS. It is a bistable toggle (on or off state) and switching to state 1 is induced by UV exposure and to state 2 is IPTG. If the toggle is set with IPTG, the cells will express cI which will inhibit Plambda. If this is exposed to certain levels of UV (see power modulations) cI is cleaved by Rec-A (a UV induced enzyme) and lacI expression begins and inhibits Ptrc.

This part improves upon its predecessor Part:BBa_K415300 in that it requires less UV power to switch states, thus killing fewer cells (see E.Coli death curve). This plasmid differs genetically from Part:BBa_K415300 in that its inhibitory lambda cI protein is more sensitive to Rec-A cleavage. We got the idea for hypersensitive cI from [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC218967/pdf/jbacter00221-0157.pdf this paper] which calls for a single point mutation in the protein. By changing the Glu233->Lys, we were able to create a toggle that is more sensitive to UV induction. We accomplished this mutation through site directed mutagenesis.

The site directed mutagenesis that the 2010 MIT iGEM team performed on the Collins toggle pTSMa in order to change it into a Low Power Toggle.

E.Coli population density as a function of UV exposure. Measured by drip assay.

Low Power Toggle switches "on" at Lower UV Power than pTSMa
Photos
This data was taken from cells co-transformed with Part:BBa_K415023 and either the Low Power Toggle (LPT) or the original toggle Part:BBa_K415300. The cells were grown to mid-log phase in LB broth and 2.65μM IPTG. The IPTG was washed out, the cells concentrated 10x, and then 100uL cells were added 1:40 to melted M9 Agar and allowed to cool in small petri dishes. The plates incubated agar up for 4 hours at 37°C, were exposed under a mask to the corresponding UV power levels, incubated for another 4 hours at 30°C and then imaged. The numbers below the images indicate the UV power level to which the cells were exposed. It is clear from the image that our LPT requires ~1/8x the UV power to switch states when compared to the original toggle.

FACS Data
FACS
This data was taken from cells co-transformed with Part:BBa_K415069 and either the Low Power Toggle or the original toggle Part:BBa_K415300. The cells were grown to mid-log phase in LB broth and 2.65μM IPTG. The IPTG was washed out, and the cells were diluted 2x in LB. 2 mL of this dilution were poured into small petri dishes and exposed with no mask at the corresponding UV power level, put into 14mL Falcon tubes and grown up for another 4 hours. Cells from each sample were added to separate tubes of 1mL PBS and FACS'd. The numbers below the images indicate the UV power level to which the cells were exposed. In each image, the x-axis is the green fluorescence measurement from our part Part:BBa_K415023 and the y-axis is the red fluorescence induced by UV exposure from the same part. We had hypothesized that the greater the UV exposure, the greater the red fluorescence (until saturation), and that the LPT would switch to the "on" state (red fluorescence) at a lower UV power than the original pTSMa.

Sequence and Features