Generator

Part:BBa_K4325027

Designed by: Laiyi Teng   Group: iGEM22_SZPT-CHINA   (2022-10-02)


pDawn-LKD-T1

Description

This composite part is a generator consisting of pDawn(cI-LVA) (BBa_K1075044), LKD (BBa_K4325004) and T1 terminator (BBa_K3033016).

Usage

The blue light responsive system pDawn(cI-LVA)(BBa_K1075044) and the lysis gene LKD (BBa_K4325004) were inserted into the pSEVA331 expression vector, which was incorporated into E. coli TOP10 and screened for bacterial colonies that grew in the dark but did not grow under blue light. Finally, the pDawn(cI-LVA)-LKD-T1 plasmid were extracted and electroporated into G. hansenii ATCC53582, after which we verified the responsiveness of pDawn(cI-LVA) to blue light.

Figure 1: Genetic circuit of pDawn(cI-LVA)-LKD-T1.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2171
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 63
    Illegal NgoMIV site found at 195
    Illegal NgoMIV site found at 289
    Illegal NgoMIV site found at 582
    Illegal NgoMIV site found at 1076
    Illegal NgoMIV site found at 1094
    Illegal NgoMIV site found at 1184
    Illegal AgeI site found at 414
    Illegal AgeI site found at 1542
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1643
    Illegal BsaI.rc site found at 525


2022 SZPT-China

Characterization

1.Batch screening of pDawn-LKD-T1 by evaluating the response to blue light.

As shown in Figure 2, except for the 16th bacterial colony, almost all of the bacteria did not grow under blue light. To further show the lysis effect of pDawn(cI-LVA)-LKD-T1, we tracked the OD 600 values of pDawn(cI-LVA)-LKD-T1 and plotted the growth curves diagram. As shown in Figure 3, E. coli TOP10-pSEVA331-pDawn(cI-LVA)-LKD-T1 is not excellent.

Figure 2: Growth situation of E. coli TOP10-pSEVA331-pDawn(cI-LVA)-LKD-T1 in the dark and under blue light.
Figure 3: Growth curves diagram of E. coli TOP10-pSEVA331-pDawn(cI-LVA)-LKD-T1 in different growth conditions.


References

[1] Robert Ohlendorf, Roee R. Vidavski, Avigdor Eldar et.al.From Dusk till Dawn: One-Plasmid Systems for Light-Regulated Gene Expression.Journal of Molecular Biology, 08 Jan 2012, 416(4):534-542.

[2] Ceyssens, P.-J. et al. Genomic analysis of Pseudomonas aeruginosa phages LKD16 and LKA1: establishment of the phiKMV subgroup within the T7 supergroup. J. Bacteriol. 188, 6924–31 (2006).

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