Difference between revisions of "Part:BBa K2277000"
 
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Lysin is a protein-coding gene. It can express a kind of lyase. This kind of lyase can crack the cell wall of E.coli cells so that intercellular products can be released to the broth. The lyase works from inside the E.coli cells and will not be released before the cell lysis.  Its lysis rate can be up to 99.8% in pET28a(+) under IPTG induction. This lyase can absolutely simplify the cells' disruption process in fermentation industry.
 
Lysin is a protein-coding gene. It can express a kind of lyase. This kind of lyase can crack the cell wall of E.coli cells so that intercellular products can be released to the broth. The lyase works from inside the E.coli cells and will not be released before the cell lysis.  Its lysis rate can be up to 99.8% in pET28a(+) under IPTG induction. This lyase can absolutely simplify the cells' disruption process in fermentation industry.
  
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===Usage and Biology===
 
  
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2277000 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2277000 SequenceAndFeatures</partinfo>
  
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<h1>Group</h1>
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ZJUT-China
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<h1>Author</h1>
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Mengyuan Chen
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<h1>Summary</h1>
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<html>
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In order to achieve cells lysis, 2017 iGEM team ZJUT-China has constructed the part BBa_K2277000. 2018 iGEM team ZJUT-China improved this part. The improved part is <a href="https://parts.igem.org/Part:BBa_K2556051">BBa_K2556051</a>.
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<br>The improvement include two aspects: 1) We improved the original basic part to a composite part, then future iGEM teams can use this part directly and use arabinose to regulate the expression of the lysin gene. 2) We added homological arms of <i>E. coli</i> genome to both ends of the part. If people want to integrate this part into the genome via CRISPR/Cas, they can directly use our part as donor DNA to facilitate their experiments. Furthermore, homological arms allowed us to insert this part into the non-metabolic pathway in <i>E. coli</i> genome, so inserting genes does not affect the normal growth of <i>E. coli</i>.
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<br>We tested BBa_K2277000 and BBa_K2556051 separately on plasmids and results are showed in Fig. 2.
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<img src="https://static.igem.org/mediawiki/2018/2/28/T--ZJUT-China--improveg.jpg" alt="" width="600px">
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<p>Fig 2. Growth curve of E. coli with original part(PL)/improved part(TL)</p>
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<br>Then we inserted our improved part into E. coli genome using CRISPR/Cas technology. In order to obtain transformants that were successfully inserted the part, we screened by plate streaking. The experimental results are showed in Fig 3.
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<img src="https://static.igem.org/mediawiki/2018/9/91/T--ZJUT-China--partl3.png" alt="" width="600px">
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<p>Fig.3 Result of plate streaking </p>
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<p>Finally, we selected 34 transformants, and 5 of them showed lysis effects on  plates containing arabinose. And one of the 5 strain showed lysis effects culturing in tubes. We named it as: E. coli MG1655-Lysis. The result are showed in Fig. 4.
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</p><img src="https://static.igem.org/mediawiki/2018/8/8e/T--ZJUT-China--partl4.png" alt="">
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<p>Fig.4 E. coli MG1655-Lysis cultured in tubes</p>
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<br>Through our improvement, the arabinose-regulated lysin gene can be more easily integrated into the genome of E. coli. Thereby reducing the number of plasmids that needed to be transformed in bacteria can also reduce the additional metabolic pressure. What’s more, it can even reduce the resistance genes carried by bacteria which are potential factors for increasing bacterial resistance. We believe that this improvement described above is exactly meaningful.
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</html>
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==Characterization from Worldshaper-HZBIOX==
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*'''Group:''' iGEM22_ Worldshaper-HZBIOX
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*'''Author:''' Yifei Hu
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*'''Summary:''' Characterization of the effect of lysin on the cell growth of E. Coli DH 5α, E. Coli MG1655 and E. Coli Nessile 1917 strains.
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====Overview ====
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The lysin gene was derived from 2017 iGEM team iGEM17_ZJUT-China. We constructed a pGLO-lysin plasmid with an arabinose inducible promoter PBAD by one-step cloning. We transformed this plasmid into E. coli DH5α, E. coli MG1655 and EcN1917. The growth of E. coli DH5α, E. coli MG1655 and EcN1917 (E. Coli Nissle 1917) treated with 30mM arabinose showed that lysin expression induced by arabinose inhibited the growth of these bacteria.
 +
====Experiments Results ====
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We constructed a pGLO-lysin plasmid with an arabinose inducible promoter PBAD using the pGLO vector and one-step cloning method. The plasmid was verified by electrophoresis and gene sequencing (Figure 1). We successfully transformed this plasmid into E. coli DH5α, E. coli MG1655 and EcN1917. DH5α were divided into three groups: DH5α group (control), DH5α+Ara(0h) group (30mM arabinose added at the beginning of culturing), and DH5α+Ara(3h) group (30mM arabinose added 3 hours after culturing). OD600 values were measured and the growth curve showed that the growth of DH5α+Ara(0h) and DH5α+Ara(3h) bacteria decreased significantly at 3h and 4h respectively (Figure 2-A). The growth curves of E. coli MG1655 treated with arabinose showed similar results (Figure 2-B).  EcN1917 transformed with pGLO-Lysin were lysed significantly by 10-hour treatment of 30mM arabinose (Figure 3-A). However, as shown in figure 3 B-D, the number of colonies of EcN1917 transformed with pGLO-Lysin was low indicating the lysin gene was expressing without arabinose induction. The reason for this result may be that pGLO belongs to a high copy number plasmid, so that the PBAD has the possibility of leaky expression. We tried to use the medium copy number pSU vector instead, but failed.
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[[File: contri-fig1.png|500px|thumb|center| Figure 1: Electrophoresis result of pGLO-lysin plasmid]]
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[[File: contri-fig2.png|600px|thumb|center| Figure 2 Effect of arabinose on the growth of E. Coli DH5α (A) and E. coli MG1655 (B) transformed with pGLO-lysin plasmid.]]
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[[File: contri-fig3.png|600px|thumb|center| Figure 3 (A) Effect of arabinose on EcN1917 transformed with pGLO-Lysin plasmid (Left: bacteria solution without arabinose, right: bacteria solution treated with 30mM arabinose for 10h); EcN1917 colonies after transformation (B: EcN1917 transformed with pGLO-Lysin plasmid, C: EcN1917 transformed with pSU plasmid, D: competent EcN1917)]]
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===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K2277000 parameters</partinfo>
 
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Latest revision as of 20:03, 30 September 2022


A gene expresses one kind of lyase which can crack the cell wall of E.coli cells.

Lysin is a protein-coding gene. It can express a kind of lyase. This kind of lyase can crack the cell wall of E.coli cells so that intercellular products can be released to the broth. The lyase works from inside the E.coli cells and will not be released before the cell lysis. Its lysis rate can be up to 99.8% in pET28a(+) under IPTG induction. This lyase can absolutely simplify the cells' disruption process in fermentation industry.


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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 555
  • 1000
    COMPATIBLE WITH RFC[1000]

Group

ZJUT-China

Author

Mengyuan Chen

Summary

In order to achieve cells lysis, 2017 iGEM team ZJUT-China has constructed the part BBa_K2277000. 2018 iGEM team ZJUT-China improved this part. The improved part is BBa_K2556051.
The improvement include two aspects: 1) We improved the original basic part to a composite part, then future iGEM teams can use this part directly and use arabinose to regulate the expression of the lysin gene. 2) We added homological arms of E. coli genome to both ends of the part. If people want to integrate this part into the genome via CRISPR/Cas, they can directly use our part as donor DNA to facilitate their experiments. Furthermore, homological arms allowed us to insert this part into the non-metabolic pathway in E. coli genome, so inserting genes does not affect the normal growth of E. coli.
We tested BBa_K2277000 and BBa_K2556051 separately on plasmids and results are showed in Fig. 2.

Fig 2. Growth curve of E. coli with original part(PL)/improved part(TL)


Then we inserted our improved part into E. coli genome using CRISPR/Cas technology. In order to obtain transformants that were successfully inserted the part, we screened by plate streaking. The experimental results are showed in Fig 3.

Fig.3 Result of plate streaking

Finally, we selected 34 transformants, and 5 of them showed lysis effects on plates containing arabinose. And one of the 5 strain showed lysis effects culturing in tubes. We named it as: E. coli MG1655-Lysis. The result are showed in Fig. 4.

Fig.4 E. coli MG1655-Lysis cultured in tubes


Through our improvement, the arabinose-regulated lysin gene can be more easily integrated into the genome of E. coli. Thereby reducing the number of plasmids that needed to be transformed in bacteria can also reduce the additional metabolic pressure. What’s more, it can even reduce the resistance genes carried by bacteria which are potential factors for increasing bacterial resistance. We believe that this improvement described above is exactly meaningful.

Characterization from Worldshaper-HZBIOX

  • Group: iGEM22_ Worldshaper-HZBIOX
  • Author: Yifei Hu
  • Summary: Characterization of the effect of lysin on the cell growth of E. Coli DH 5α, E. Coli MG1655 and E. Coli Nessile 1917 strains.

Overview

The lysin gene was derived from 2017 iGEM team iGEM17_ZJUT-China. We constructed a pGLO-lysin plasmid with an arabinose inducible promoter PBAD by one-step cloning. We transformed this plasmid into E. coli DH5α, E. coli MG1655 and EcN1917. The growth of E. coli DH5α, E. coli MG1655 and EcN1917 (E. Coli Nissle 1917) treated with 30mM arabinose showed that lysin expression induced by arabinose inhibited the growth of these bacteria.

Experiments Results

We constructed a pGLO-lysin plasmid with an arabinose inducible promoter PBAD using the pGLO vector and one-step cloning method. The plasmid was verified by electrophoresis and gene sequencing (Figure 1). We successfully transformed this plasmid into E. coli DH5α, E. coli MG1655 and EcN1917. DH5α were divided into three groups: DH5α group (control), DH5α+Ara(0h) group (30mM arabinose added at the beginning of culturing), and DH5α+Ara(3h) group (30mM arabinose added 3 hours after culturing). OD600 values were measured and the growth curve showed that the growth of DH5α+Ara(0h) and DH5α+Ara(3h) bacteria decreased significantly at 3h and 4h respectively (Figure 2-A). The growth curves of E. coli MG1655 treated with arabinose showed similar results (Figure 2-B). EcN1917 transformed with pGLO-Lysin were lysed significantly by 10-hour treatment of 30mM arabinose (Figure 3-A). However, as shown in figure 3 B-D, the number of colonies of EcN1917 transformed with pGLO-Lysin was low indicating the lysin gene was expressing without arabinose induction. The reason for this result may be that pGLO belongs to a high copy number plasmid, so that the PBAD has the possibility of leaky expression. We tried to use the medium copy number pSU vector instead, but failed.

Figure 1: Electrophoresis result of pGLO-lysin plasmid
Figure 2 Effect of arabinose on the growth of E. Coli DH5α (A) and E. coli MG1655 (B) transformed with pGLO-lysin plasmid.
Figure 3 (A) Effect of arabinose on EcN1917 transformed with pGLO-Lysin plasmid (Left: bacteria solution without arabinose, right: bacteria solution treated with 30mM arabinose for 10h); EcN1917 colonies after transformation (B: EcN1917 transformed with pGLO-Lysin plasmid, C: EcN1917 transformed with pSU plasmid, D: competent EcN1917)

Functional Parameters