ShanghaiTech iGEM

Characterization

We have characterized the BBa_K2152004 from 2016 ShanghaitechChina_B which is a kill switch. (http://parts.igem.org/Part:BBa_K2152004) We utilized this part as a kill switch in our project. (For more detail, visit https://2019.igem.org/Team:ShanghaiTech_China/Safety).

Validation

We have submitted three parts as our new parts.

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. Both the expression of our target protein and molecule are validated by SDS-Page and mass spectrum respectively.
For more details, visit NAS page.

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 target gene in a short time after inducement.
For more details, visit BBa_3278001.

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 higher promotion effect on FixJ expression. In this part, we aim to mitigate leakage and make a more reliable blue light control system.
For more details, visit BBa_3278006.

Improvement

Tip: The part BBa_3278002 is for improvement in judging form.
The part BBa_3278006 is one of our new parts.

Expression leakage and delay are the two main defects problems of pDawn system.

Leakage refers to the expression of target protein without inducement of light. According to our measurement, we discovered that although the ssamples are striclystrictly kept away from light, there is still had significant a great expression of GFP after being cultured for one night. Since we employ this system to control the production of NAS to moderate blood sugar level, such large leakage is not allowed here.

Therefore, we carefully studied the gene circuits of pdawn system [2] [3] and hypothesized that enhancing the strength of the promoter of YF1 may enhance the repression of protein expression in the dark, thus solving the leakage problem. Also, we considered to change the sequence of YF1 domain to promote the activity of this enzyme to realize leakage mitigation.

Here is the circuit of pDawn:

Part I: Changing promoter

To do so, We selected The four robust promoters, T7, tac, J23119, and J23100 are chosen, which are relative robust promoters. The four promoters are synthesized by bio company with two overlapping sequences with the target vector on the two ends. Next, we used Gibson assembly cloning kit to recombine the promterpromoter fragments and the target vector to obtain new vectors.

pDawn with T7 promoter is abbreviated as T7c below. pDawn with tac promoter is abbreviated as tac below. pDawn with modified tac promoter is abbreviated as mtac below. pDawn with J23119 promoter is abbreviated as 119 below. pDawn with J23100 promoter is abbreviated as 100 below.

The PCR bands one in the red frame is the pcr product ofare the linearized vector, with the theoretical length of 7770 bps. The marker on the left is 8.000 K DNA Marker.

The bands in the red framefrom the colony PCRs are the products of clony PCR.shown above before sequencing confirmed.

Here are the sequencing result:

For T7c:See this part as BBa_3278001

For tac:See this part as BBa_3278005

For mtac:See this part as BBa_3278002

For 119:See this part as BBa_3278003

For 100:See this part as BBa_3278004

Here is the final plasmid:

Part II: Changing YF1

At the same time, we tried to change YF1 into YF2. YF2 is reported in Andreas Möglich’s paper[2]. By changing 24 bps in the original sequence, YF2 was reported to have higher promotion effect on FixJ expression, which means stronger inhibitory effect on target gene’s expression.

Amino acid sequence of YF1 & YF2:

YF2 has advantage in cutting down leakage:

So we used PCR to change the 24bp segment.After the transformation, we did colony PCR and verified the correct size of the plasmid which is 7213bps:

And we sequenced the modified plasmid and verified the correct sequence of our modified plasmid:See this part as BBa_3278006

REFERENCES

[1] From Dusk till Dawn: One-Plasmid Systems for Light-Regulated Gene Expression. Robert Ohlendorf1, Roee R. Vidavski, Avigdor Eldar, Keith Moffat, and Andreas Möglich. Journal of Molecular Biology, 2012, Vol.416, pp. 534-542.

[2] Design and Signaling Mechanism of Light-Regulated Histidine Kinases. Andreas Möglich, Rebecca A. Ayers and Keith Moffat. Biophysical Journal, 2009, Vol.96 (3), pp. 1433-1444.