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Team:Peking/secondtry/Suicide
From 2014.igem.org
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).
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, Plac (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.
Results
Efficiency test of our kill switch
We transformed the two plasmids (Fig.3) into E.coli, where holin is expressed under the inducible promoter Plac while endolysin under a library of constitutive promoters. Then, a gradient of concentration of inducer was applied and the growth rate was measured. Compared with the bacteria carrying blank plasmids, the efficiency of our kill switch could be evaluated.
These data above show that the OD600 of the stationary-phase E. coli expressing holin and endolysin is lower than that carrying blank plasmids, which indicates that our kill switch just inhibits the growth of E. coli to some extent but does not lead to cell lysis.
We thought the poor compatibility of the two plasmids in our kill switch is the main cause of our failure in experiment. Hence, to solve this problem, we designed a new kill switch where the transcription unit for endolysin and that for holin is inserted into the same plasmid (Fig.5), and we will test its efficiency in our future work.
Reference
[1]Bieʼnkowska-Szewczyk, K., Lipiʼnska, B., & Taylor, A. (1981). The R gene product of bacteriophage λ 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, λ 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.
