Team:NEU CHINA/Improve

PARTS_Improve Biobrick

PARTS

In the second approach, we designed a protease-based post-translational degradation regulation method [3]. We integrated a protein degradation tag sequence AAV next to the reporter gene and reduced the output basal expression. To reduce the background expression without sacrificing the high output, we next incorporated the sensor into a TEV protease-based reporter protein degradation control system (Fig. 3). This integrated regulation system was sufficient to reduce the sensor’s leakage while also being able to maintain both the sensor’s output amplitude and sensitivity, leading to expanded output dynamic range. However, due to the time limitation, we would perfect this part after iGEM.

REFERENCES

[1]Lin, H.Y., P.J. Bledsoe, and V. Stewart, Activation of yeaR-yoaG operon transcription by the nitrate-responsive regulator NarL is independent of oxygen- responsive regulator Fnr in Escherichia coli K-12. J Bacteriol, 2007. 189(21): p. 7539-48.

[2]Merulla, D. and J.R. van der Meer, Regulatable and Modulable Background Expression Control in Prokaryotic Synthetic Circuits by Auxiliary Repressor Binding Sites. ACS Synth Biol, 2016. 5(1): p. 36-45.

[3]Fernandez-Rodriguez, J. and C.A. Voigt, Post-translational control of genetic circuits using Potyvirus proteases. Nucleic Acids Res, 2016. 44(13): p. 6493-502.

[4]Aizenman, E., H. Engelberg-Kulka, and G. Glaser, An Escherichia coli chromosomal "addiction module" regulated by guanosine [corrected] 3',5'-bispyrophosphate: a model for programmed bacterial cell death. Proc Natl Acad Sci U S A, 1996. 93(12): p. 6059-63.

[5]Pandey, D.P. and K. Gerdes, Toxin-antitoxin loci are highly abundant in free-living but lost from host-associated prokaryotes. Nucleic Acids Res, 2005. 33(3): p. 966-76.

[6]Amitai, S., Y. Yassin, and H. Engelberg-Kulka, MazF-mediated cell death in Escherichia coli: a point of no return. J Bacteriol, 2004. 186(24): p. 8295-300.

[7]Stirling, F., et al., Rational Design of Evolutionarily Stable Microbial Kill Switches. Mol Cell, 2018. 72(2): p. 395.

Figure 1. Diagram for NO sensor system in pCDFDuet-1. PyeaR indicated the promoter region that senses to NO. Both of Native NsrRBS and Extra NsrRBS are the NsrR binding sequences. The reporter gene is Luciferase and highlighted in green.

Last year, the NO sensor (BBa_K2817000:NorR-pnorv-amplicp) based expression system with a serious gene leakage problem. At first, we considered the NorR over expression might be the reason of gene leakage. However, after we knocked out the norR gene, the leakage even more increased (Fig. 4B), and it seems that the reporter gene expression was NO sensor independent. We speculated that the plasmid constructed last year lacked a terminator downstream the norR sequence. Therefore, we integrated one terminator B0010/B0012 sequence to the inflammation sensor (Fig. 4A) and the amilCP leakage problem has been significantly alleviated (Fig. 4C).

Figure 3. Tuning the sensor background and output dynamic range via reporter degradation regulation. Schematic showed protease-mediated regulation of the background and output dynamic range for a NO sensor. ‘A’ represents the AAV degradation tag. Off state: without NO induction. On state: with NO induction.

Figure 4B. Pellets of bacteria transformed with constructed NO sensor plasmid after 4 hours’ induction at 37℃. From left to right: control, 0.5mM IPTG without SNP, 1mM IPTG without SNP, 100μM SNP,0.5mM IPTG with 100μM SNP, 1mM IPTG with 100μM SNP. From top to bottom: empty vector, T7-NorR-PnorV-amplicp, T7-PnorV-amplicp.

Figure 4C. Pellets of bacteria transformed with constructed NO sensor plasmid after 2hours’ induction at 37 ℃. From left to right: control, 0.5mM IPTG without SNP, 1mM IPTG without SNP, 0.5mM IPTG with 100μM SNP, 1mM IPTG with 100μM SNP. From top to bottom: empty vector, T7-NorR-PnorV-amplicp, T7-T-PnorV-amplicp.

Figure 2. The response to NO sensors. A. The response to NO of PyeaR-luc in ECN. Histogram of Luminescence (RLU): empty vector, PyeaR-luc without SNP, empty vector, PyeaR-luc with 100μM SNP. B. Comparison genetic leakage expression of PytfE-luc and PyeaR-NsrRBS-luc systems with or without NO induction. Blue bars indicated the luciferase expression percent under the NO induction, while Red bars showed the percentage of genetic leakage without NO induction.

The improvement of NO sensors

This year, we chose BBa_K2967017 (PyeaR-Luc) as an alternative to our inflammatory sensor, due to its sensitivity to nitrate and nitrite. When nitrate and nitrite enter into E. coli, they will be converted to nitric oxide. Then nitric oxide will bind to the repressor protein NsrR that inactivates PyeaR to inhibit the downstream gene transcription [1].

However, we noticed detectable gene expression leakage from the characterization of the NO sensor (PyeaR-Luc) (Fig. 2A). To reduce sensor’s too much leakage, we intended to utilize two different approaches. For the first approach, we inserted an extra NsrR binding sequence (NsrRBS) at the downstream of PyeaR to create a ‘road-blocking’ effect [2] (Fig.1). Comparing to the unmodified PyeaR-Luc system (Fig.2B), the histogram of luminescence data demonstrated that the relative lower luciferase signal in PyeaR-NsrRBS system in the absence of NO.

The improvement of 'kill switch'

Last year, we constructed the engineered bacteria transformed with PcspA-mazF(BBa_K2817006) plasmid as the “kill switch” to eliminated any possible bio-hazard outcomes. However, the survival rate of PcspA-mazF transformed E. coli was significantly low at 37 °C (Fig.5). We suspected the high toxicity of MazF directly resulted in bacterial death and the “kill switch” need to be optimized.

Figure 5. The effect of the last year’s kill switch based on PcspA-mazF. The effect of empty vector and PcspA-mazF on the growth of E. coli BL21 at 37 °C.

This year, we introduced the toxin-antitoxin system-mazEF (Fig 6.), a natural toxin-antitoxin system found in E. coli, to soothe the serious leakage problem of mazF in order to optimize the effect of kill switch. MazF is a stable toxin protein, while MazE is an unstable antitoxin protein [4]. When the bacterial growth are compromised by environmental stress and the expression of mazEF is inhibited, the unstable antitoxin protein is preferentially degraded, and the relative content of the stable toxin protein increases to a certain extent, which will lead to the death of bacterial [5,6]. The mazEF expression is regulated by the cold-acting promoter cspA that can only be efficiently activated at a low temperature of, for example, 16 °C [7].

Firstly, we constructed two new plasmids, one was pet28a with mazE as target gene, the other was pCold with mazF as target gene. Then, we co-transformed the two plasmids, PT7-mazE and PcspA-mazF, into E. coli BL21 competent cells. After diluting to $OD_{600}$=0.02 on the second day, the cells were cultured at 16 °C (Fig. 7A) or 37 °C (Fig. 7B) and the survival rate was measured every hour for constitutive 11 hours. Finally, the growth curve of two temperatures was plotted.

The survival rate of cells with mazE-mazF system at 37°C around 88.06% (Fig 3). In comparison with the survival rate of cells with PcspA-mazF plasmid, the cells transformed with PT7-mazE and PcspA-mazF plasmids showed highly significant improvement in terms of the survival rate of cells. Therefore, the utilization of MazE did substantially reduce the indexes of the necrosis of engineered bacteria and the introduction of mazEF system could significantly to reduce the toxicity of MazF and enhance the efficiency of kill switch.

Improve Biobrick

Figure 6. Kill switch module construction.A. The construction of PcspA-mazF plasmid. PcspA, the cold shock promoter. mazF, the toxin gene from E. coli.B. The construction of T7promoter-mazE plasmid. mazE, the antitoxin gene from E. coli.

Figure 4A. Diagram for NO sensor system in pCDFDuet-1. T7 promoter, the gene downstream of this promoter will be transcribed when there is T7 RNA polymerase. lacO, the sequence represses the nearby promoter when there is no inducer (e.g. IPTG). RBS, ribosome binding site. NorR, NO binding protein. PnorV, a promoter which is sensitive to NO. amilCP, blue chromoprotein.

Figure 7. The effect of our new kill switch under different conditions. A, B. The effect of empty vector, mazF and co-transformed mazE-mazF system on the growth of E.coli BL21 at different temperatures (A. 37°C; B. 16°C).