Team:Peking/Suicide

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<p>The <b>Fig. 5(a)</b> shows that the difference between the OD<sub>595nm</sub> of experimental group and control group is obvious in the late logarithmic phase. The <b>Fig. 5(b)</b> shows that the growth curve of the <i>E. coli</i> 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<sub>595nm</sub>’s difference. Hence, the OD<sub>595nm</sub>’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.</p>
<p>The <b>Fig. 5(a)</b> shows that the difference between the OD<sub>595nm</sub> of experimental group and control group is obvious in the late logarithmic phase. The <b>Fig. 5(b)</b> shows that the growth curve of the <i>E. coli</i> 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<sub>595nm</sub>’s difference. Hence, the OD<sub>595nm</sub>’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.</p>
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<p>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<sub>lac</sub>, we will construct a plasmid where the expression of GFP is under control of P<sub>lac</sub> 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 <i>E. coli</i>. </p>
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<p>In this experiment, none of tested groups entered 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 was that the expression of holin and endolysin is not enough to cause cell lysis. Hence, in order to test the performance of P<sub>lac</sub>, we will construct a plasmid where the expression of GFP is under control of P<sub>lac</sub> and measure the fluorescence intensity after the addition of a gradient of IPTG. According to the results above, we found an appropriate concentration of IPTG to get better results. Besides, the relatively low toxicity of holin and endolysin may be another cause and we can choose the <i>Ccd</i>A/<i>Ccd</i>B 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 <i>E. coli</i>. </p>
<h2>References</h2>
<h2>References</h2>

Revision as of 03:31, 18 October 2014

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).

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 membranes. However, this inhibition process is subverted by the dissipation of proton motive force and in this case, S107 would 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 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 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 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, Plac, 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.

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 into pSB1C3. Endolysin is expressed under a constitutive promoter, BBa_J23106 and holin is expressed under an inducible promoter, Plac.

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 Plac 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.

Figure 5. The growth curves of the E. coli carrying suicide switch and blank plasmid. X axis is the culture time and we get OD595nm value every five minutes. Y axis is the OD595nm of E. coli. 10mM IPTG was added in experimental group (red line) while none of IPTG was added in control group (blue line). The surrounding light-colored lines are error bars. (a) The growth curves of E. coli carrying suicide switch. (b) The growth curves of E. coli carrying blank plasmid.

The Fig. 5(a) shows that the difference between the OD595nm 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 OD595nm’s difference. Hence, the OD595nm’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, none of tested groups entered 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 was 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. According to the results above, we found an appropriate concentration of IPTG to get better results. 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.

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