Difference between revisions of "Part:BBa K3927002"
Line 19: Line 19:
  
 
===Characterization===
 
===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.
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Revision as of 18:11, 14 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


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 203
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 185
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 203
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 203
  • 1000
    COMPATIBLE WITH RFC[1000]

Description

Usage

Design

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.