Part:BBa_K4832004
KC5
In order to ensure the biosafety of our engineered bacteria, we introduced LacI operons into plasmids that can express the Plagiodera versicolora actin protein dsRNA to regulate the expression of dsRNA. In addition, on the basis of the introduction of LacI operons, we successfully integrated the KillerRed protein expression frame to construct the KC5 plasmid, which further ensured the safety of our construction of engineered bacteria.
Usage and Biology
LacI operon construction
Considering that the expression of plasmid dsRNA is constitutive, in order to control its expression, we introduced LacI operator at one end of the tac promoter and transformed it into an inducible dsRNA expression plasmid. The expression of dsRNA is regulated by IPTG induction.
Figure 1.KC3 plasmid map
KillerRed
Our team is particularly focused on biosafety, and after introducing the LacI operon, we decided to add a kill switch - KillerRed.
The KillerRed protein expressed by the KillerRed gene changes its structure after being stimulated by a certain amount of light, causing cells to produce highly toxic oxygen free radicals, thus achieving the purpose of killing cells.
To this end, we further successfully integrated the KillerRed protein expression frame on the basis of the vector KC3, and successfully constructed the KC5 plasmid, which further ensured the safety of our engineered bacteria.
Figure 2. KC5 plasmid map
Characterization
LacI operon
The KC3 plasmid was introduced into P.chlororaphis KC-P, and then actin sequence was used as the probe for northern blot verification, as shown in Figure 2.
Figure 3. Northern BlotFirst laneP.chlororaphis KC-P+KC3+IPTG， third lane P.chlororaphis KC-P+KC3。
According to Figure 3, LacI operator can significantly inhibit the expression of actin-dsRNA. However, P. viridis KC-P+KC3 without IPTG induction still showed a small amount of color.
We introduced KC5 plasmid into P.chlororaphis KC-P and verified it. As shown in Figure 4, the number of growing colonies on the plate with 1h illumination was less than 5% of that on the plate with 0h illumination, indicating that our plasmid had a good lethal effect.
Figure 4.In order, the plate image after illumination is 0h, 0.5h, 1h, and 2h.
We also model it and describe it with ordinary differential equation.
Figure 5.N is the number of viable bacteria, t represents the light time, and k is the mortality rate of bacteria
The ode45 function in MATLAB is used to solve the differential equation, and the result is compared with the actual data, and the error is calculated. Then, the fminsearch function is used to fit the unknown parameter k to minimize the error. Since there may be multiple local optimal solutions in the fitting results, multiple fitting is carried out to find the global optimal solution. According to the fitting results, the differential equation is re-solved, the final model is obtained, and the fitting curve is drawn, as shown in the figure. The fitting results show that the value of parameter k is 0.07
Index fitting:
MATLAB curve fitting toolbox was used to fit the existing data. By comparing the R2 and root mean square error (RMSE) of different approximation results, it was found that the best fitting effect was achieved by using exponential approximation. The revised R2 was as high as 97.03% and the RMSE was 5.883, and the final fitting curve expression was as follows
Figure 6.Our data analysis modeling of the KillerRed kill switch.
The model is the same as the logarithmic residual model of the high-temperature sterilization bacteria. From the experimental results and the model prediction results, it can be seen that after the introduction of KillerRed, only a short time of light can kill the vast majority of bacteria, but it does not completely kill almost all bacteria like the high-temperature sterilization. This also shows that compared with the physical killing method, the biological suicide switch caused the death of bacteria is relatively mild, but it is enough to meet our biological security needs.
Reference
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[2]Narang A, Oehler S. Effector Overlap between the lac and mel Operons of Escherichia coli: Induction of the mel Operon with β-Galactosides. J Bacteriol. 2017;199(9):e00796-16. Published 2017 Apr 11. doi:10.1128/JB.00796-16
[3]Santillán M, Mackey MC. Quantitative approaches to the study of bistability in the lac operon of Escherichia coli. J R Soc Interface. 2008;5 Suppl 1(Suppl 1):S29-S39. doi:10.1098/rsif.2008.0086.focus
[4]Zander D, Samaga D, Straube R, Bettenbrock K. Bistability and Nonmonotonic Induction of the lac Operon in the Natural Lactose Uptake System. Biophys J. 2017;112(9):1984-1996. doi:10.1016/j.bpj.2017.03.038
[5]Marbach A, Bettenbrock K. lac operon induction in Escherichia coli: Systematic comparison of IPTG and TMG induction and influence of the transacetylase LacA. J Biotechnol. 2012;157(1):82-88. doi:10.1016/j.jbiotec.2011.10.009
[6]Bulina ME, Chudakov DM, Britanova OV, et al. A genetically encoded photosensitizer. Nat Biotechnol. 2006;24(1):95-99. doi:10.1038/nbt1175
[7]Liu X, Shi R, Zou D, et al. Positive selection vector using the KillerRed gene. Anal Biochem. 2011;412(1):120-122. doi:10.1016/j.ab.2011.01.034
[8]Waldeck W, Heidenreich E, Mueller G, Wiessler M, Tóth K, Braun K. ROS-mediated killing efficiency with visible light of bacteria carrying different red fluorochrome proteins. J Photochem Photobiol B. 2012;109:28-33. doi:10.1016/j.jphotobiol.2012.01.002
[9]Onukwufor JO, Trewin AJ, Baran TM, Almast A, Foster TH, Wojtovich AP. Quantification of reactive oxygen species production by the red fluorescent proteins KillerRed, SuperNova and mCherry. Free Radic Biol Med. 2020;147:1-7. doi:10.1016/j.freeradbiomed.2019.12.008
[10]Trewin AJ, Berry BJ, Wei AY, Bahr LL, Foster TH, Wojtovich AP. Light-induced oxidant production by fluorescent proteins. Free Radic Biol Med. 2018;128:157-164. doi:10.1016/j.freeradbiomed.2018.02.002
[11]Li X, Fang F, Gao Y, et al. ROS Induced by KillerRed Targeting Mitochondria (mtKR) Enhances Apoptosis Caused by Radiation via Cyt c/Caspase-3 Pathway. Oxid Med Cell Longev. 2019;2019:4528616. Published 2019 Mar 7. doi:10.1155/2019/4528616
[12]Shibuya T, Tsujimoto Y. Deleterious effects of mitochondrial ROS generated by KillerRed photodynamic action in human cell lines and C. elegans. J Photochem Photobiol B. 2012;117:1-12. doi:10.1016/j.jphotobiol.2012.08.005
[13]Takemoto K, Matsuda T, Sakai N, et al. SuperNova, a monomeric photosensitizing fluorescent protein for chromophore-assisted light inactivation. Sci Rep. 2013;3:2629. doi:10.1038ep02629
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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