Difference between revisions of "Part:BBa K1378031"
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<p>We transformed the two plasmids into <i>E.coli</i>. 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.</p>
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<p>We transformed the two plasmids into <i>E.coli</i>. Then, 10mM of inducer was applied and the growth rate was measured. Compared with the bacteria carrying blank plasmid, the efficiency of our suicide switch can be evaluated.</p>
  
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<figure><img src="https://static.igem.org/mediawiki/2014/f/fa/Peking2014zsy_data.png"><figcaption><b>Figure 5. </b> <b>The growth curves of the <i>E. coli</i> carrying suicide switch and blank plasmid. </b>X axis is the culture time and we get OD<sub>595nm</sub> value every five minutes. Y axis is the OD<sub>595nm</sub> of <i>E. coli</i>. 1mM IPTG was added in experimental group <b>(blue line)</b> while none of IPTG was added in control group <b>(red line)</b>. (a) The growth curves of <i>E. coli</i> carrying suicide switch. We have repeated this experiment for three times and the surrounding light-colored lines are error bars. (b) The growth curves of <i>E. coli</i> carrying blank plasmid of the first-time experiment. (c) The growth curves of <i>E. coli</i> carrying blank plasmid of the second-time experiment.</figcaption></figure>
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<figure><img src="https://static.igem.org/mediawiki/2014/f/fa/Peking2014zsy_data.png"><figcaption><b>Figure 5. </b> <b>The growth curves of the <i>E. coli</i> carrying suicide switch and blank plasmid. </b>X axis is the culture time and we get OD<sub>595nm</sub> value every five minutes. Y axis is the OD<sub>595nm</sub> of <i>E. coli</i>. 10mM IPTG was added in experimental group <b>(red line)</b> while none of IPTG was added in control group <b>(blue line)</b>. The surrounding light-colored lines are error bars. <b>(a)</b> The growth curves of <i>E. coli</i> carrying suicide switch. <b>(b)</b> The growth curves of <i>E. coli</i> carrying blank plasmid. </figcaption></figure>
 
   
 
   
  
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<p>The Fig. 5(a) shows that the difference between the OD<sub>595nm</sub> 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<sub>595nm</sub> is that the <i>E. coil</i> have entered decline phase. The Fig. 5(b) and Fig. 5(c) show that the growth curves of the <i>E.coli</i> 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<sub>595nm</sub>’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<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>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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=='''Reference'''==
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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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=='''References'''==
 
<p>[1]Gründling, A., Bläsi, U., & Young, R. (2000). Biochemical and genetic evidence for three transmembrane domains in the class I holin, lambda <i>S</i>. <i>Journal of Biological Chemistry, 275</i>(2), 769-776.</p>
 
<p>[1]Gründling, A., Bläsi, U., & Young, R. (2000). Biochemical and genetic evidence for three transmembrane domains in the class I holin, lambda <i>S</i>. <i>Journal of Biological Chemistry, 275</i>(2), 769-776.</p>
 
<p>[2]Young, R., Wang, I. N., & Roof, W. D. (2000). Phages will out: strategies of host cell lysis. <i>Trends in microbiology, 8</i>(3), 120-128.</p>
 
<p>[2]Young, R., Wang, I. N., & Roof, W. D. (2000). Phages will out: strategies of host cell lysis. <i>Trends in microbiology, 8</i>(3), 120-128.</p>

Revision as of 02:51, 18 October 2014

Holin from lambda phage

Holin is a 105-amino-acid-residue cytoplasmic membrane protein with three transmembrane domains, naturally expressed by double-stranded lambada phage. Holin will oligomerize and form a hole on the inner membrane of host bacteria at a certain time at an allele-specific time. And then the formation of hole will help Endolysin, a kind of lysozyme, come out from cytoplasm to periplasm to degrade peptidoglycan and inhibit the respiration by eliminating proton gradient.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

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)[1]. 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[2]. Additionally, as its name suggests, S107 can bind to S105 and inhibit its function specifically[3]. 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[4]. 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.

Characterization

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).

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.

We transformed the two plasmids into E.coli. Then, 10mM of inducer was applied 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. 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, 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 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 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 E. coli.

References

<p>[1]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.

[2]Young, R., Wang, I. N., & Roof, W. D. (2000). Phages will out: strategies of host cell lysis. Trends in microbiology, 8(3), 120-128.

[3]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.

[4]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.