Results

Engineering of the GshF expression and GSH production module

The goal of this module is to construct an expression system of the bifunctional glutathione synthetase GshF, which can be employed to enhance glutathione production in G. hansenii. To this end, the gene gshF was firstly optimized regarding the codon sequence, and then ligated to pSEVA331, which was finally introduced into E. coli TOP10 to obtain the expression system. To the DNA constructs, specific primers were designed to amplify a partial region of gshF, and the size of the PCR product met with our expectation (Figure 1a). In addition, to examine the efficacy of the expression system, the molecular weight of the HiS-tag-labelled GshF protein was checked using western blot assay (Figure 1b). Interestingly, even in the genetic background of E. coli TOP10, GshF is active and functional since the GSH production has been significantly increased in TOP10 following transformation with the pSEVA311-based gshF expression system (Figure 1c).

Engineering of the lysis and safety module

This module was slightly modified from the module from our last year project, the objective of which is to construct a light-inducible system for cell lysis. To this end, the lysis genes originated from bacteriophages were combined with the blue light responsive system pDawn, so that illumination with blue light could induce the lysis gene expression and, in turn the bacterial cell death. In this way, the glutathione produced by the GshF expression system as well as cell lysate can also be released to exert cosmetic effect as active ingredients. Moreover, this could also act as a kill switch which enables confinement of an engineered bacterium to an environment without blue light.

Table 1: The library of the constructed kill switches

Kill Switch	Part Component
BX01	pDawn(cI-LVA)-X174E-T1
BX02	pDawn(cI-LVA)-RBS070-X174E-T1
BX03	pDawn(cI)-X174E-T1
BX04	pDawn(cI-LVA)-X174E-LVA-T1
BL01	pDawn(cI-LVA)-LKD-T1
BL02	pDawn(cI-LVA)-RBS070-LKD-T1
BL03	pDawn(cI)-RBS070-LKD-T1
BL04	pDawn(cI-LVA)-RBS070-LKD-LVA-T1

A small library of potential constructs were built to screen for effective kill switches (Table1). The agarose gel electrophoresis verified that the four genetic builds (BX01, BX02, BL01, BL02) in E. coli TOP10 were correct (Figure 3).

Then, the strains that grew in the dark but failed to grow under blue light (red squares in Figure 4) were selected and cultured in fresh LB medium and the growth curves of these strains were monitored under the two different illumination conditions (Figure 5). The discrepancy in OD₆₀₀ value in the dark and that under blue light was used to evaluate the efficacy of the blue light inducible system. We found that BX01 and BL02 were the optimal candidates. We found that BX01 and BL02 were the optimal candidates.

However, even for the most effective system BL02, the growth-limiting effect under blue light could only be maintained for several hours. Moreover, following growth in the dark until the logarithmic phase, blue light illumination did not even inhibit bacterial growth. These results indicated that this blue light-responsive suicide system become dysfunctional over a period of growth. We speculated that excessive expression of lysis protein under blue light and leakage expression of lysis protein in the dark might cause a survival pressure. Consequently, favorable mutants outcompeted the parental strains and thrived in the culture broth. Therefore, we intended to optimize both BX01 and BL02.

The kill switch variants BX03, BX04, BL03 and BL04 were therefore generated either by deleting the degradation tag LVA in the original repressor cI of pDawn or adding the degradation tag LVA downstream the lysis gene. The plasmids were introduced into E. coli TOP10 by electroporation to obtain the transformants (Table 1). Then, the E. coli strains that grew in the dark but not under blue light were selected (BX03, BL03, BL04 in Figure 7) and cultured in LB medium in the dark until the OD₆₀₀ reached around 0.6. Finally, the bacterial growth was further monitored under blue light. We found that those three new engineered strains showed decreased OD₆₀₀ values in response to blue light (Figure 8), indicating the effective refinement of the inducible suicide plasmid in rapid growing cells.

Assembly of GshF expression module and lysis module

Table 2: The library of the Recombinant Plasmid for GSH production and release

Recombinant Plasmid	Part Component
GX01	pSEVA331-pDawn(cI-LVA)-X174E-gshF-T1
GX03	pSEVA331-pDawn(cI)-X174E-gshF-T1
GL02	pSEVA331-pDawn(cI-LVA)-RBS070-LKD-gshF-T1
GL03	pSEVA331-pDawn(cI)-RBS070-LKD-gshF-T1
GL04	pSEVA331-pDawn(cI-LVA)-RBS070-LKD-LVA-gshF-T1

The objective was to incorporate the lysis-induction circuit into the pSEVA311-based gshF expression system. To this end, the inducible suicide genetic circuit covering the pDawn promoter, the lysis gene and the associated regulatory region was amplified by high-fidelity PCR. Then the PCR product was purified and ligated to linearized the gshF expression system vector to generate a set of assembled systems (Table 2). Those plasmids were individually introduced into E. coli TOP10 competent cells, which were further spread on selective plates to obtain transformants. The assembly of two modules in each type of transformant was verified by PCR (Figure 9).

To examine the efficacy of the inserted lysis modules in E. coli,the growth of those strains on agar surface under either the dark or the blue light condition was compared, and the result allowed us to confirm the insertion of optogenetic circuit for lysis in the colonies of those strains (Figure 10 and 11). Among them, the plasmid, GL03 was optimal since the frequency of E. coli colonies harboring this plasmid that can lyse under blue light was the highest.

Prototype

See proof of concept for details.

References

[1] https://2021.igem.org/Team:SZPT-CHINA/Design
[2] Li, W., Li, Z., Yang, J. & Ye, Q. Production of glutathione using a bifunctional enzyme encoded by gshF from Streptococcus thermophilus expressed in Escherichia coli. J. Biotechnol.154, 261–268 (2011).
[3] Teh, M. Y. et al.An Expanded Synthetic Biology Toolkit for Gene Expression Control in Acetobacteraceae. Acs Synth. Biol.8, 708–723 (2019).
[4] Pophaly, S. D. et al.Glutathione biosynthesis and activity of dependent enzymes in food-grade lactic acid bacteria harbouring multidomain bifunctional fusion gene (gshF). J. Appl. Microbiol.123, 194–203 (2017).
[5] Ryu, M.-H. & Gomelsky, M. Near-infrared Light Responsive Synthetic c-di-GMP Module for Optogenetic Applications. ACS Synth. Biol.3, 802–810 (2014).
[6] Florea, M. et al.Engineering control of bacterial cellulose production using a genetic toolkit and a new cellulose-producing strain. Proc. Natl. Acad. Sci. U. S. A.113, E3431–E3440 (2016).
[7] Ohlendorf, R., Vidavski, R. R., Eldar, A., Moffat, K. & Möglich, A. From Dusk till Dawn: One-Plasmid Systems for Light-Regulated Gene Expression. J. Mol. Biol.416, 534–542 (2012).
[8] Witte, A., Wanner, G., Sulzner, M. & Lubitz, W. Dynamics of PhiX174 protein E-mediated lysis of Escherichia coli. Arch. Microbiol.157, 381–8 (1992).
[9] Ceyssens, P.-J. et al.Genomic analysis of Pseudomonas aeruginosa phages LKD16 and LKA1: establishment of the phiKMV subgroup within the T7 supergroup. J. Bacteriol.188, 6924–31 (2006).
[10] Guiziou, S. et al.A part toolbox to tune genetic expression in Bacillus subtilis. Nucleic Acids Res.44, 7495–7508 (2016).
[11] Stirling, F. et al.Rational Design of Evolutionarily Stable Microbial Kill Switches. Mol. Cell68, 686-697.e3 (2017).