Team:ShanghaiTech China/Improvement

ShanghaiTech iGEM

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 problems of pDawn system.

Leakage refers to the expression of target protein without light induction. According to our measurement, we discovered that although the samples are strickly kept away from light, there is still a significant expression of GFP after being cultured for one night. Since we employed this system to control the production of NAS to moderate blood sugar levels, 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 before YF1 may enhance the repression of target protein in the dark, thus solving the leakage problem. Also, we considered changing 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 four robust promoters, T7, tac, J23119, and J23100 are chosen, which are relatively robust promoters. The four promoters are synthesized by bio company with two overlapping sequences (same with the target vector on the two ends). Next, we used the Gibson assembly cloning kit to recombine the promoter fragments and the target vector to obtain new plasmids.

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 bands in the red frame are the PCR of the linearized vectors, with the theoretical length of 7770 bps. The marker on the left is 8,000 K DNA Marker.

The bands in the red frame are the products of colony PCR. Band #1, #2, #4 and #5 are the expected products and are sequenced subsequently.

Here are the sequencing results:

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:

For 100:

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 a higher promotion effect on FixJ expression, which means the 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

Results

For mtac and YF2:

We compared their performances with the original pDawn-mEGFP component based on both the efficiency of mEGFP expression and the leakage level. To examine mEGFP expression dynamics, different illuminating duration was used in our experiment.

To see detailed Micrographs, go to the specific part pages listed above.

To see the protocol of our characterization, go to Light Control.

We compared YF2, mtac with pDawn-mEGFP as below.

Figure 3: Average F.I. line graphs of pDawn-mEGFP, mtac, YF2, tac normalized based on duration = 0 h. The detailed methods of data analysis and normalizing are shown at the bottom of this part.

Result: Comparing with the original pDawn-mEGFP, a similar effect can be observed in YF2 and mtac. To quantify the expression efficiency and leakage level, further analysis was carried out as below.

The effect on reducing leakage level was proven with the following graphs.

Figure 4: Average F.I. bar graphs of pDawn-mEGFP, mtac, YF2, tac in separate timepoints normalized based on duration = 0 h. Timepoints were chosen based on the tendency of the line graph with 12h as the rapid growth period, 24h as the development period and 30h, 36h as the stable period.

Figure 5: Bar graphs of average F.I. in light-off group of pDawn-mEGFP, mtac, YF2, tac at the timepoints of 12h, 24h, 30h, 36h after normalization based on the original pDawn-mEGFP groups, characterizing the leakage level of the plasmids

Result: Figures above clearly show the decreased background expression level of mtac and YF2 compared with pDawn-mEGFP. Specifically, the YF2 maintained a relatively low level of leakage which also dipped over time, while mtac showed a downward trend after 12h before becoming stable after 30h. The lowest leakage of the mtac and YF2 occurred at 12h and 36h respectively, with mtac reducing the leakage by 78.74% and YF2 by 78.98% when compared with pDawn-mEGFP.

Expression efficiency was compared in the following graphs:

Figure 6: Bar graphs of relative F.I. with the fluorescence level of the light-on group divided by dark group of pDawn-mEGFP, mtac, YF2, tac at the timepoints of 12h, 24h, 30h, 36h, characterizing the expression efficiency of the system.

Result: Comparing with the original pDawn-mEGFP, a similar expression efficiency can be observed in YF2 and mtac and the timepoint of 12h and 36h witnessed a substantial climb of the YF2 and mtac respectively, exceeding pDawn-mEGFP by approximately 50%.
As a result, mtac and YF2 can be characterized as the most ideal modified components among all of the candidates, with similar efficiency as the original one and better performance on leakage reducing. Though the expression level of the 2 modified components is not yet ideal enough, we can expect a potential more excellent result obtained under suitably higher light intensity or a more proper inducing environment, which will be our plan for the future.

For T7c:

Firstly, we quantified the absolute expression level of the T7c.

Figure 2: Average Absolute F.I. line graphs of pDawn-mEGFP, T7c, tac, showing the absolute expression capacity

Result: A significant increase of the absolute expression level compared to the original pDawn-mEGFP can be observed directly from the graph in the first several timepoints, indicating T7c’s excellent property of rapid reaction with time.

T7c highlighted a significant increase of the absolute expression level compared to the original pDawn-mEGFP, reacting rapidly with a short time range, which can be further used as a more sensitive and rapid component for light-control. As for the leakage problem, we expect a combination of T7c with other low leakage modified components such as YF2 in order to weaken the leakage while obtaining a relatively high expression level.

To see more details, go to the websites below.
origin pDawn: BBa_K1075044
pDawn with T7 promoter: BBa_K3278001
pDawn with mtac promoter: BBa_K3278002
pDawn with tac promoter: BBa_K3278005
pDawn with YF2: BBa_K3278006

References

[1] Ohlendorf, R., Vidavski, R. R., Eldar, A., Moffat, K. & Moglich, A. From dusk till dawn: one-plasmid systems for light-regulated gene expression. J Mol Biol 416, 534-542, doi:10.1016/j.jmb.2012.01.001 (2012).

[2] Moglich, A., Ayers, R. A. & Moffat, K. Design and signaling mechanism of light-regulated histidine kinases. J Mol Biol 385, 1433-1444, doi:10.1016/j.jmb.2008.12.017 (2009).

[3] Reyrat, J. M., David, M., Blonski, C., Boistard, P. & Batut, J. Oxygen-regulated in vitro transcription of Rhizobium meliloti nifA and fixK genes. J Bacteriol 175, 6867-6872, doi:10.1128/jb.175.21.6867-6872.1993 (1993).