pDawn-RBS070-LKD-T1

Description

This composite part is a generator containing pDawn (cI-LVA) (BBa_K1075044) and LKD (BBa_K4325004).

Usage

The pDawn (cI-LVA) blue light response system (BBa_K1075044) and lysis gene LKD (BBa_K4325004) were inserted into the pSEVA331 expression vector, which was inserted into E. coli TOP10, screened out the bacterial colony which grew in the dark but did not grow under blue light. Finally, the pDawn-RBS070-LKD-T1 plasmid was selected and inserted into G. hansenii ATCC53582 by electroporation to verify the responsiveness of pDawn (cI-LVA) under blue light.

Figure 1:

Gene circuit of pDawn(cI-LVA)-RBS070-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(cI-LVA)-RBS070-LKD-T1 in response to blue light lysis in E. coli TOP10.

As shown in Figure 2,except for the 11^th and 13^th bacterial colony,almost all of the bacterial colony did not grow under blue light. To further explore the lysis effect of pDawn (cI-LVA) -RBS070-LKD-T1-TOP10, we measured the OD ₆₀₀ values of pDawn (cI-LVA)-RBS070-LKD-T1-TOP10 and ploting the growth curve diagram. As shown in Figure 3, the pDawn (cI-LVA)-RBS070-LKD-T1-TOP10 lysis effect was well in the first eight hours, but was unstable after fifteen hours.

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

Figure 3: Growth curve diagram of pDawn(cI-LVA)-RBS070-LKD-T1-TOP10

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.