Part:BBa_K1697002
Toggle switch for gram positive
Moved from BBa_K1697001
this part was designed with gram positive bacteria in mind. However, we are unable to characterize it in gram positive organism in E. coli
Design
https://parts.igem.org/File:Toggle_Switch_Activity_Assay.jpg
Usage and Biology
The promoter in the system are Phyperspank(Phs) and PxylA. Phyperspank is a constitutive promoter for gram positive when the repressor protein, LacI is not present. When present, LacI bound to Phs, inhibiting the expression of the promoter. However, with addition of lactose or IPTG, LacI would bind to IPTG and detach from the promoter, causing the promoter to express the protein downstream. PxylA is another constitutive promoter for gram positive. PxylA is repressed by xylR. Xylose would bind to xylR and cause PxylA to express the protein downstream. We express xylR under the effect of Phs and express LacI under the effect of PxylA. Thus, when one promoter is active, it repress the other promoter. When PxylA is active, it express SboA, the spermicidal protein. It also express LacI which inhibit the other promoter, Phs. When induced with IPTG, LacI binds to IPTG and inhibition on Phs decrease. Thus protein downstream of Phs active, which is XylR. XylR repress PxylA, thus the expression of GFP stop, and the production of RFP started.
Method: To characterize the toggle switch, we swtich the SboA with GFP and NdoA with RFP. Single colony was streaked over LB agar with antibiotic. After 18 hours, the culture was induced with 1M IPTG and observed under UV.
Result: induction of culture with IPTG and incubation of 18 hours create distinguishable green fluorescence compared to wild type and uninduced
image 1 upper plate: induced with IPTG lower left: not induced with IPTG lower right: wild type
however, induction with xylose did not show any change in color, qualitatively. We have tried inducing colonies never been induced by IPTG before, or colonies that have been induced by IPTG before. Both showed no significant change in color. Thus, we try to transform RFP to see if BL21 CP do really express red colored RFP in the culture. The transformed culture with pSB1C3 showed no red fluoresecence. Thus we hypothesize that the toggle switch work as intended. however, further investigation with more sophisticated device is needed.
https://parts.igem.org/File:IMG_20150919_133731.jpg
figure 2 show difference between pOXGW-toggle in BL21 CP and pSB1C3 in TOP10 top left: BL21 CP, pOXGW-toggle, induced with IPTG: showing green fluorescence top right: TOP10, pSB1C3-toggle, not induced with IPTG, showing green fluoresence bottom left: Wild type BL21CP, not showing fluorescence bottom right: WIld type TOP10, not showing fluorescence https://parts.igem.org/File:IMG_20150919_134417.jpg
Conclusion: we hypothesize it is working as intended, however, further approach concerning detecting RFP expression on BL21 CP is needed.
https://parts.igem.org/File:Toggle_Switch_Activity_Assay.jpg.png
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 2042
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 3777
Illegal AgeI site found at 3889 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1836
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