Part:BBa_K3927002
3C120-CYC-LacO
This part encodes for a truncated CYCp core promoter with three C120 repeats replacing the native upstream activating sequence, and a lacO sequence downstream of the TATA box.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 203
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 185
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 203
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 203
- 1000COMPATIBLE WITH RFC[1000]
Description
This part is an improvement for part BBa_K3927001 (C120-CYC promoter), and aims to improve overall induction fold when induced under blue light while reducing promoter leakiness.
2 additional C120 repeats have been added onto BBa_K3927001 (C120-CYC promoter) upstream of the truncated CYCp core promoter with intent to increase overall induction fold of the promoter in the presence of blue light. A LacO sequence has been added downstream of the TATA box with the goal of providing repression of the inherent promoter leakiness in the absence of blue light.
Usage
Design
Presented with the dilemma of increasing overall expression while preventing increased leakiness, an abstracted construct of a modular promoter was developed. Modelling for promoter activation kinetics showed that simply increasing the activiy of activation elements is likely to be accompanied by an increase in leakiness. This modular promoter would consist of a core promoter and an upstream activation module that would increase activity in the presence of blue light, similar to the construction of the C120-CYC basic promoter. However, this design includes an additional repression module, that is meant to be repressed in the in darkness to suppress leakiness. In the presence of blue light, this module should be de-repressed, allowing the activated promoter to function as per normal. Thus, it would require two repression modules, one to directly repress the promoter of interest, and a second blue light activated repressor to repress the primary repression module. With this design, it was hoped that strong blue light activation modules with rapid activation kinetics could be developed without the pitfalls of increased leakiness
Characterization
This part was characterized by placing the part upstream of a mKO reporter gene. The overall performance of the composite part was quantified based on the levels of mKO produced.
A DNA fragment containing the C120-CYC promoter, as well as a constitutive expression cassette for NLS-VP16-EL222 was ordered from IDT, and Gibson assembly was used to assemble it into a plasmid with the mKO orange fluorescent protein terminated by LSC2 terminator, producing the plasmid pC120-mKO-EL-U. The plasmid was then transformed into BY4741 to test for EL222 mediated blue light induction of mKO.
pC120-mKO-EL-U(BY4741) was compared to the wildtype BY4741 as a negative control. Cells were cultured overnight, and the next day two cultures inoculated in 25ml YNB-URA media to OD600~1.2, and then cultured in a shaking incubator at 30 degrees Celsius, 220rpm in either blue light or darkness, with wildtype BY4741 undergoing an identical protocol. After 6 hours, the cells were washed and the level of mKO fluorescence(Ex:515, Em:560) was measured and normalized to the OD of the culture to ascertain the level of mKO expression under the C120-CYC promoter.
Other than the absolute induction fold, it was also important to characterize the effect of dose depended activation as well as the possible metabolic burden that the circuit may impose. As such, the experiment was replicated with the same induction protocol, but instead of an endpoint measurement the OD600 and mKO expression was measured hourly to plot the expression and growth curve in darkness, 100% blue light or using a 50%, half-hour-on half hour-off cycle.
| None |