Coding

Part:BBa_K2277000

Designed by: Geyi Wang   Group: iGEM17_ZJUT-China   (2017-10-17)


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.

Functional Parameters

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Categories
Parameters
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