Team:Peking/Suicide

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Introduction

Our project aims at eliminating algae 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 algae’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).

Figure 1. The model for the membrane topology of S107 and S105. S105 consist of three transmembrane domains (TMD) with an N-out, C-in topology while S107 only has two TMDs, caused by an extra positive charge conferred by Lys2. The S107 can inhibit the function of S105, preventing it from forming holes in cell membrane. However, this inhibition can be subverted by the dissipation of proton motive force and in this case, S107 will become active holin, accelerating the rate of pore formation.

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).

Figure 2. Model for export and activation of λ phage endolysin. In λ phage, the holin is inserted in cell membrane without forming holes and endolysin is restricted within cytoplasm (Cyt) before 'lysis time'. However, at an allele-specific time, the holin oligomerizes to form holes in CM, allowing endolysin to reach and hydrolyze PG, leading to cell lysis.

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 Plac for its high operability.

Figure 3. Model for detecting AHLs produced by M. aeruginosa. The AHLs produced by M. aeruginosa can diffuse into the cytoplasm of E. coli and then bind the TraR to activate the expression of target genes, turning the suicide switch.

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, Plac, 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.

Figure 4. The final construct of killing switch. The transcription unit that expresses endolysin is inserted into the plasmid pSB1A2 and that for holin is inserted to pSB1C3. Endolysin is expressed under a constitutive promoter and holin is expressed under an inducible promoter, Plac.

Result

Efficiency test of our suicide switch

We transformed the two plasmids (Fig.4) into E.coli, where holin is expressed under the inducible promoter Plac 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.

Figure 5. The growth curves of the E. coli carrying suicide switch and blank plasmid after the addition of 1mM of IPTG or none. After the addition of IPTG, the E. coli carrying suicide switch grew slower and its OD595 in stationary phase is obviously lower relative to that without addition of inducer. However, the growth curve of the E. coli carrying blank plasmid with addition of 1mM of IPTG is nearly coincident with that without addition of IPTG.

This figure above shows that our suicide switch is detrimental to the growth of E. coli to some extent but does not eradicate the host after induction.

We thought 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 Plac, we will construct a plasmid where the expression of GFP is under control of Plac 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.

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