pDawn-RBS070-LKD-LVA-T1

Description

This composite part is a generator consisting of pDawn(cI-LVA)(BBa_K1075044),RBS070(BBa_K4325002), LKD-LVA (BBa_K4325011)and T1 terminator(BBa_K3257021).

Usage

We inserted the pDawn(cI-LVA) blue light response system (BBa_K1075044) and the lysis gene with hydrolysis tag LKD-LVA (BBa_K4325011) 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 plasmid containing pDawn (cI-LVA)-LKD-LVA-T1 were introduced into G. hansenii ATCC53582 by electroporation to verify the responsiveness of pDawn(cI-LVA) to blue light.

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(cI-LVA)-LKD-LVA-T1 in response to blue light lysis in E. coli.

As shown in Figure 1, the plasmid containing pDawn(cI-LVA)-RBS070-LKD-LVA-T1 was introduced into E. coli TOP10 and performed the agarose gel electrophoresis. Then we performed the drop plate assay in the dark and under blue light. After 12 hours, we found that the 4^th, 5^th, 6^th, 14^th bacterial colonies grew in the dark and did not grow under blue light, (Figure 2) which indicates that pDawn(cI-LVA)-RBS070-LKD-LVA-T1 functions as intended in .coli. E

Figure 1: (1) pSEVA331-pDawn(cI)-X174E-T1; (2) pSEVA331-pDawn(cI-LVA)-X174E-LVA-T1; (3) pSEVA331-pDawn(cI)-LKD-T1; (4) pSEVA331-pDawn(cI-LVA)-RBS070-LKD-LVA-T1 were constructed successfully.

Figure 2:Growth condition of pDawn(cI-LVA)-RBS070-LKD-LVA-T1-TOP10 in the dark and under the light

References

[1]Ohlendorf, R., Vidavski, R. R., Eldar, A., Moffat, K. & Möglich, A. From Dusk till Dawn: One-Plasmid Systems for Light-Regulated Gene Expression. J. Mol. Biol. 416, 534-542 (2012).

[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).