pDawn-RBS070-X174E-T1

Description

This composite part is a generator consisting of pDawn(cI-LVA)(BBa_K1075044) and X174E(BBa_K1835500).

Usage

The pDawn (cI-LVA)(BBa_K1075044) blue light response system and the lysis gene X174E(BBa_K1835500) were inserted into the pSEVA331 expression vector,which wsa inserted into the E.coli TOP10,screened out bacterial colony that grew in the dark but did not grow under blue light. Finally, the pSEVA331-pDawn-RBS070-X174E-T1 plasmid was selected and inserted into G. hansenii ATCC53582 to verify the responsiveness of pDawn(cI-LVA) to blue light.

Figure 1:Gene circuit of pDawn(cI-LVA)-RBS070-X174E-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 pSEVA331-pDawn(cI-LVA)-RBS070-X174E-T1 in response to blue light lysis in E. coli.

As shown in Figure 2, the 11^th and 15 ^th bacterial colony did not grow under blue light but grow in dark. In order to further explore the lysis effect of pSEVA331-pDawn (cI-LVA)-RBS070-X174E-T1-TOP10, we measured the OD₆₀₀ value of pSEVA331-pDawn (cI-LVA)-RBS070-X174E-T1-TOP10 periodically andploting the growth curve diagram.As shown in Figure 3, the lysis effect of pSEVA331-pDawn (cI-LVA)-RBS070-X174E-T1-TOP10 is not well.

Figure 2:

The growth condition of pDawn(cI-LVA)-RBS070-X174E-T1-TOP10 in the dark and under the blue light.

Figure 3:

Growth curve diagram of pDawn(cI-LVA)-RBS070-X174E-T1-TOP10.

2.pSEVA331-pDawn(cI-LVA)-RBS070-X174E-T1 in response to blue photolysis in G. hansenii ATCC53582.

pDawn(cI-LVA)-RBS070-X174E-T1 was inserted into G. hansenii ATCC53582 by electroporation and identified. As shown in Figure 4, pDawn(cI-LVA)-RBS070-X174E-T1 was successfully inserted into G. hansenii ATCC53582.