pDawn-B0034-X174 E-rrnB T1

Description

This composite part is a generator consisting of the pDawn promoter and X174 E.

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

2021 SZPT-China

Biology

This composite part is a generator consisting of the pDawn (BBa_K1075044) promoter and X174 E (BBa_K1835500).

Usage

The target genes of pDawn blue light response system (BBa_K1075044) and X174 E (BBa_K1835500) were inserted into the pSEVA331 expression vector. Then, random primer guided mutagenesis method to modulate the strength of the RBS (BBa_B0034) located upstream of X174 E. Finally, the pDawn-RBSNNN-X174 E-rrnB T1 (BBa_K3740044) plasmid was constructed and introduced into E. coli. E. coli isolates that grow normally in the dark and cannot grow under blue light were screened out. Finally, the plasmid was extracted, and further introduced into G. hansenii ATCC 53582, in which the responsiveness of pDawn to blue light was also verified.

Figure 1. Gene circuit of pDawn-RBSNNN-X174 E-rrnB T1.

Characterization

1. Batch screening of pSEVA331-pDawn-RBSNNN-X174 E-rrnB T1- pSEVA331in response to blue light lysis in E. coli.

Method: We use the random primer method to modulate the strength of the RBS (BBa_B0034) located upstream of X174 E. After introducing into E. coli DH5α, a drop plate assay was performed to screen the bacterial isolate that can grow normally in the dark but cannot under the blue light irradiation.

As shown in Figure 11, the 4th isolate that grew normally in the dark but did not under blue light, indicating that we successfully expressed the cleavage protein X174 E in E. coli.