Composite

Part:BBa_K1695038

Designed by: Ma Lee   Group: iGEM15_CityU_HK   (2015-08-13)


RBS Bacteriophage λ Codon Optimized Lysis Cassette S mutant R Rz


The gene cassette encodes S protein (holin), R protein (endolysin) and Rz protein (spanin) from bacteriophage λ. All three genes in the cassette are codon optimized and paired with the strong RBS BBa_B0034. The S gene contains a missense mutation such that alanine (A) at position 50 is replaced by glycine (G). The mutation was introduced to shorten the lysis time.


Results and Characterization

Upon induction with 0.2 mM, 1 mM and 5 mM of IPTG, recombinant E. coli harboring the lysis cassette Smutλ-Rλ-Rzλ (BBa_1695038) showed a sharp drop in absorbance (A600) after 15 minutes whereas cells without IPTG induction showed continued as indicated by the steady increase in OD (Figure 1A). The results show that 0.2 mM IPTG is sufficient to initiate cell lysis after 15 minutes, and the speed of cell lysis is not increased at higher concentrations of IPTG (Figure 1B). Overall, the use of (1) OD600 measurement to determine cell growth or cell lysis (panel A) and (2) plate count (cfu; panel B) showed that the Lysis Gene Cassette is induced by IPTG and resulted in the initiation of cell lysis after 15 minutes of IPTG induction. Cells were completely lysed by 20 minutes. It would be interesting in future studies to determine if the time of cell lysis could be regulated by IPTG at concentrations below 0.2 mM.

2015CityU HK BBa K1695038 Characterization.png

Figure 1. Cell lysis upon induction of lysis cassette by IPTG in E. coli cells

Recombinant E. coli carrying lysis cassette Smutλ-Rλ-Rzλ (BBa_1695038) was cultured in minimal medium (supplemented with 0.2% glucose and 0.5% casamino acid). IPTG at various concentrations (0 mM, BLUE) (0.2 mM, ORANGE), 1 mM, GREY) (5 mM, YELLOW) was added to the bacterial culture at OD600 ~ 0.6. Samples were taken at 5-min and 10-min intervals from IPTG induced and uninduced cultures for (A) OD600 measurements and (B) cell plating on LB solid medium (CFU count) to determine the percentage (%) of cell survival, respectively.


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 AgeI site found at 546
  • 1000
    COMPATIBLE WITH RFC[1000]


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