Team:Peking/secondtry/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 level of biosafety in 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 and would be switched on only in appropriate condition. Our results show these genes are controlled strictly therefore rendering a nearly harmless phenotype to engineered bacteria. Once be induced, however, they can also function efficiently to eradicate their host.

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 kill switch based on the λ lysis model. The S holin, or called S₁₀₅, encoded by S gene, a dual-start motif of λ phage, is a 105-amino-acid-residue CM protein with three transmembrane domains (TMD)^[3]. S₁₀₇, or 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 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 TMD, 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).

We use holin and endolysin for our suicide system. In our design, endolysin is controlled by a constitutive promoter while holin by inducible promoter, P_lac (Fig. 3). During the killing and degrading process, expression of holin is repressed, 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 restoring the aquatic ecosystem.

**Figure 3.** The final construct of kill switch plasmid BBa_k1378033. One transcription unit that expresses endolysin and one that expresses holin are inserted into the same plasmid. Endolysin is expressed under a constitutive promoter and holin is expressed under an inducible promoter, P_lac.

Additionally, we designed two tests for our kill switch. These are toxicity tests with endolysin and holin to estimate whether either of them can cause cell lysis alone (Fig.4). Detailed information will be described in the Results.

**Figure 4.** The plasmids for testing the toxicity of endolysin and holin. (a) BBa_k1378031: Endolysin is expressed a constitutive promoter and this transcription unit is inserted into the plasmid pSB1C3. (b) BBa_k1378032: Holin is expressed under an inducible promoter, P_lac and this transcription unit is inserted into the plasmid pSB1C3.

Results

Toxicity test of endolysin

First, we tested whether the endolysin would lead to cell lysis without the presence of holin. In our design, endolysin is expressed under a constitutive promoter (Fig. 4a). The bacteria carrying this plasmid were cultured and their growth rate was measured. Compared with the bacteria carrying blank plasmid, the influence could be evaluated.

Toxicity test of holin

Next, for evaluating the necessity of applying endolysin into our suicide system and the toxicity of holin, it was tested whether and how fast the holin could cause cell death without the help of endolysin. In this design, holin is expressed under the inducible promoter P_lac (Fig. 4b). A gradient of concentration of inducer was applied and growth rate of bacteria was measured, regarded as criteria to quantify the suicide effect.

Efficiency test of our kill switch

At last, we transformed the plasmid BBa_k1378033 (Fig. 3) into E.coli, where holin is expressed under the inducible promoter P_lac while endolysin under a library of constitutive promoters. As well, a gradient of concentration of inducer was applied and the growth rate was measured. Compared with the bacteria carrying the plasmid BBa_k1378032 and the ones carrying blank plasmid, the efficiency of our kill switch could be evaluated.

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, &#955 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.