Difference between revisions of "Part:BBa K3927002"

Revision as of 01:39, 18 October 2021

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

Description

3C120-CYC-LacO is the implementation of an abstracted, hypothetical synthetic promoter developed by the NUS iGEM team 2021 for tight, blue light regulated expression in S.cerevisiae.

Usage

This part is a blue light inducible promoter, therefore requires blue light for this promoter to be induced. Additionally, it requires part BBa_K3927000 / BBa_K3570021 (NLS-VP16-EL222) and part BBa_K3927006 (yeast LacI) to function.

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 activity 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.