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, are 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 are expressed by 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 show these genes render a nearly harmless phenotype to engineered bacteria and once be induced, they can inhibit the growth of host to some extent.

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 design 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 behavior. Like many other bacteria, Microcystis aeruginosa also produce 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 plan to use TraR as a sensor to turn the suicide switch in our final design, but at this stage, we choose the inducible promoter P_lac for its high operability.

Circuit Design

We choose λ lysis system to construct suicide switch due to its high efficiency and natural occurrence, and we introduce 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 inducible promoter, P_lac, because high concentration of holin can cause cell death alone (Fig. 4). During the killing and degradation process, expression of holin is repressed by the lacI in the genome, thus restricting endolysin within cytoplasm and keeping host alive. After completion of work, however, expression of holin will be derepressed by the addition of IPTG and holin will oligomerize in CM to form a few large holes that release the trapped endolysin into periplasm. Endolysin in periplasm will attack PG and then cause lysis of the host. As a result, our transgenic E. coli will be eradicated finally, thus avoiding polluting the environment. Additionally, if the lethality of holin is too strong, we can apply antiholin in our suicide switch.

Results

Efficiency test of our suicide switch

We transformed the two plasmids (Fig.4) into E.coli, where holin is expressed under the inducible promoter P_lac while endolysin under the constitutive promoter. Then, 1mM of inducer was applied empirically and the growth rate was measured. Compared with the bacteria carrying blank plasmid, the efficiency of our suicide switch can be 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, indicating that our suicide switch can inhibit the growth of E. coli. A possible explanation for the decrease in OD_595nm is that the E. coil have entered decline phase. The Fig. 5(b) and Fig. 5(c) show that the growth curves of the E.coli carrying blank plasmid after the addition of 1mM IPTG are nearnly coincident with that without addition of IPTG, excluding the possibility that the toxicity of IPTG leads to the noticeable OD_595nm’s difference. The difference between the pattern in Fig. 5(b) and Fig. 5(c) may be caused by the different culture environment. However, both the curves in Fig. 5(b) and Fig. 5(c) have consistency, and thus our experiment should be repeatable. 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 our experiment, we think the quite low OD_595nm value in stationary phase and early coming of decline phase are closely relative to the growth activity of E. coil, but it does not obstruct us from drawing a right conclusion. For better results, we will select E. coil with better growth activity to do the experiment again. 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. 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.

Reference

[1]Bieʼnkowska-Szewczyk, K., Lipiʼnska, B., & Taylor, A. (1981). The R gene product of bacteriophage &#955 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.