Team:Peking/Suicide

Introduction

Our project aims at eliminating cyanobacteria and recovering aquatic ecosystem. Because of this, after completing killing and degradation work, it is necessary to clear away all of remaining engineered Escherichia coli in natural water. This measure also prevents leakage of foreign genes, which improves the biosafety level of our project.

To realize our design, two kinds of protein from λ bacteriophage, holin and endolysin, were chosen for their high lethality to E. coli. Holin is a membrane protein which can oligomerize to form holes in cytoplasmic membrane (CM). Endolysin is a murein transglycosylase^[1]. It is able to cross the CM to attack the peptidoglycan (PG) with the help of holes formed by holins, thus leading to cell lysis^[2].

These suicide genes mentioned above were expressed under a set of inducible promoters (e.g. promoters which can be induced by cyanobacteria’s quorum sensing signals) and would be switched on only in appropriate conditions. Our results showed these genes render a nearly harmless phenotype to engineered bacteria and once be they could inhibit the growth of host to some extent once induced.

Design

Holin

Holin is a generic term to describe a group of small proteins produced by double-stranded DNA bacteriophage to trigger holes formation at the end of lytic cycle. In our project, we designed our suicide switch based on the λ lysis model. The S holin, also called S₁₀₅, encoded by S gene, a dual-start motif of λ phage, is an 105-amino-acid-residue CM protein with three transmembrane domains (TMD)^[3]. S₁₀₇, also called antiholin, is the other protein encoded by S gene, differing from the S holin only by the Met-Lys N-terminal extension. However, this difference confers to S₁₀₇ an extra positive charge, which prevents its TMD1 from inserting into the CM^[4]. Additionally, as its name suggests, S₁₀₇ can bind to S105 and inhibit its function specifically^[5]. In λ lysis system, S₁₀₇ and S₁₀₅ are encoded by S gene at ratio of approximately 1:2, which is defined by the two RNA structure, and if the amount of S₁₀₇ is increased relative to S₁₀₅, the 'lysis time' will be delayed^[6]. The inhibition function of S₁₀₇ can be subverted by collapsing proton motive force, which also allow insertion of TMD1 of S₁₀₇ into CM, instantly increasing the amount of active holin by making previously inactive S₁₀₇ - S₁₀₅ complexes functional (Fig. 1).

Endolysin

The λ phage endolysin is an 18-kDa soluble protein with murein transglycosylase activity^[1]. In λ lysis system, enzymatically active endolysin accumulate in cytoplasm without harm to host bacteria before 'lysis time' because the holin accumulate in CM without disturbing its integrity during this time. However, at an allele-specific time, the holin oligomerizes to form a small number of large holes, allowing the endolysin to cross the CM and attack the PG ^[2][7] (Fig. 2).

Acyl-homoserine lactones

Acyl-homoserine lactones, or AHLs for short, are a class of signaling molecules involved in bacterial quorum sensing, which enable the coordination of group-based behaviour. Like many other microbes, Microcystis.aeruginosa also produces AHLs, which is important for their biofilm formation^[8]. In addition, these signals produced by M. aeruginosa can serve as ligands for TraR^[8], a cytoplasmic receptor as well as a transcription activator from Agrobacterium.tumefaciens^[9], and make the TraR-AHL complex stable to activate the transcription of downstream expression. Hence, we planned to use TraR as a sensor to turn the suicide switch in our final design, but at this stage, we chose the inducible promoter P_lac for its high operability.

Circuit Design

We chose λ lysis system to construct suicide switch due to its high efficiency and natural occurrence, and we introduced both endolysin and holin because of their cooperativity in cell lysis, which improves the performance of our suicide switch. In our design, endolysin is controlled by a constitutive promoter while holin by an inducible promoter, P_lac, because accumulation of holin can cause cell death alone (Fig. 4). During the killing and degradation process, expression of holin is repressed by the lacI in genome, thus restricting endolysin within cytoplasm and keeping host alive. After completion of work, however, expression of holin is derepressed by the addition of IPTG and holin will oligomerize in CM to form large holes allowing the trapped endolysin exporting into periplasm. Endolysin in periplasm would attack PG and then cause lysis of the host. As a result, our transgenic E. coli were designed to be eradicated at last, thus avoiding contanminating environment. Additionally, if the lethality of holin is too strong, antiholin could be applied in suicide switch.

Results

Efficiency test of our suicide switch

We transformed these two plasmids (Fig.4) into E. coli, where holin was expressed under the inducible promoter P_lac while endolysin under the constitutive promoter, BBa_J23106. Then, 10mM of inducer was applied and the growth rate was measured. Compared with the bacteria carrying blank plasmids, the efficiency of our suicide switch was evaluated.

The Fig. 5(a) shows that the difference between the OD_595nm of experimental group and control group is obvious in the late logarithmic phase. The Fig. 5(b) shows that the growth curve of the E. coli carrying blank plasmid after the addition of 10mM IPTG is nearly coincident with that without addition of IPTG, excluding the possibility that the toxicity of IPTG leads to the noticeable OD_595nm’s difference. Hence, the OD_595nm’s difference should be caused by the slowed growth rate or cell death, and combining the working mechanism of holin and endolysin, we believe the cell death may be the main cause and our suicide switch may have some bactericidal effect.

In this experiment, every group did not enter the stationary phase and we thought better results could be attained by prolonging culture time. One possible reason for this performance of our suicide switch is that the expression of holin and endolysin is not enough to cause cell lysis. Hence, in order to test the performance of P_lac, we will construct a plasmid where the expression of GFP is under control of P_lac and measure the fluorescence intensity after the addition of a gradient of IPTG. According to the results of the experiment above, we will find an appropriate concentration of IPTG to get a better result if necessary. Besides, the relatively low toxicity of holin and endolysin may be another cause and we can choose the CcdA/CcdB Type II Toxin-antitoxin system instead in our future work because it have been proved that the CcdB, a topoisomerase poison targeting the GyrA subunit of DNA gyrase, shows strong toxicity to E. coli.

References

[1]Bieʼnkowska-Szewczyk, K., Lipiʼnska, B., & Taylor, A. (1981). The R gene product of bacteriophage lambda is the murein transglycosylase. Molecular and General Genetics MGG, 184(1), 111-114.

[2]Wang, I. N., Smith, D. L., & Young, R. (2000). Holins: the protein clocks of bacteriophage infections. Annual Reviews in Microbiology, 54(1), 799-825.

[3]Gründling, A., Bläsi, U., & Young, R. (2000). Biochemical and genetic evidence for three transmembrane domains in the class I holin, lambda S. Journal of Biological Chemistry, 275(2), 769-776.

[4]Young, R., Wang, I. N., & Roof, W. D. (2000). Phages will out: strategies of host cell lysis. Trends in microbiology, 8(3), 120-128.

[5]Bläsi, U., Chang, C. Y., Zagotta, M. T., Nam, K. B., & Young, R. (1990). The lethal lambda S gene encodes its own inhibitor. The EMBO journal, 9(4), 981.

[6]Bläsi, U., Nam, K., Hartz, D., Gold, L., & Young, R. (1989). Dual translational initiation sites control function of the lambda S gene. The EMBO journal, 8(11), 3501.

[7]Dewey, J. S., Savva, C. G., White, R. L., Vitha, S., Holzenburg, A., & Young, R. (2010). Micron-scale holes terminate the phage infection cycle. Proceedings of the National Academy of Sciences, 107(5), 2219-2223.

[8]Zhai, C., Zhang, P., Shen, F., Zhou, C., & Liu, C. (2012). Does Microcystis aeruginosa have quorum sensing?. FEMS microbiology letters, 336(1), 38-44.

[9]Fuqua, W. C., & Winans, S. C. (1994). A LuxR-LuxI type regulatory system activates Agrobacterium Ti plasmid conjugal transfer in the presence of a plant tumor metabolite. Journal of bacteriology, 176(10), 2796-2806.