Team:ShanghaiTech China/Validation

ShanghaiTech iGEM

We have submitted three parts as our new parts.

pDawn-NAS

The first one is pDawn-NAS, which combines pDawn system and N-acyl synthase (NAS) genes together, trying to modulate the synthesis of N-acyl amine with blue light. The expression of our target molecule is validated by mass spectrum respectively.

Fig1(a). LC-MS result of control and experiment groups

Fig1(b). The fragmentation peak of pDawn-NAS E. coli BL21:DE3

Fig1(c). The fragmentation peak of standard N-palmitoyl serinol Figure 1:The LC-MS results showed that the target molecule can be normally secreted, which proved that the NAS gene can work well under the regulation of pDawn.

For more details, visit BBa_3278007 and NAS page.

pDawn with YF2

The Third one is the pDawn system with the important histidine kinase changed from YF1 to its homologous protein YF2, which is reported to have a higher promotion effect on FixJ expression. In this part, we aim to mitigate leakage and make a more reliable blue light control system.

Fig3(a). pDawn-YF2-0 hour

Fig3(b). pDawn-YF2-6 hour

Fig3(c). pDawn-YF2-12 hour

Fig3(d). pDawn-YF2-24 hour

Fig3(e). pDawn-YF2-30 hour

Fig3(f). pDawn-YF2-36 hour Figure 1:Representative images of YF2 expression under different light illumination-durations. The groups labeled ‘light-on’ were under light induction, while controls labeled ‘light-off’ were kept in the dark wrapped with aluminum foil. pictures were taken with the same parameters as the Alexa Fluor 488 dye, with the same 1000-ms exposure time for all images. The Brightfield images were captured with 300-ms exposure time.
Result: Comparing with the original pDawn-mEGFP, YF2 featured an ideal expression capacity and a minimum leakage level.

For more details, visit BBa_3278006.
For the protocol of characterization, visit Light Control.

pDawn with T7 promoter

The second one is the pDawn system with the promoter in front of YF1 and FixJ changed from lacIq to T7 promoter. In this part, we try to optimize the expression of the target gene in a short time after inducement.

Fig2(a). pDawn-T7c-0 hour

Fig2(b). pDawn-T7c-6 hour

Fig2(c). pDawn-T7c-12 hour

Fig2(d). pDawn-T7c-24 hour

Fig2(e). pDawn-T7c-30 hour

Fig2(f). pDawn-T7c-36 hour Figure 2: Representative images of T7c under different light illumination-durations. The groups labeled ‘light-on’ were under light induction, while controls labeled ‘light-off’ were kept in the dark wrapped with aluminum foil. pictures were taken with the same parameters as the Alexa Fluor 488 dye, with the same 1000-ms exposure time for all images. The Brightfield images were captured with 300-ms exposure time.
Result: For T7c, the absolute fluorescence intensity reached a relatively high level in a short time, namely reacting faster to light induction, despite the fact that the leakage level was not reduced.

For more details, visit BBa_3278001.
For the protocol of characterization, visit Light Control.