pDawn-RBS070-LKD-T1

Description

This composite part is a generator containing pDawn(cI-LVA) (BBa_K1075044), RBS070 (BBa_K4325002), LKD (BBa_K4325004) and T1 terminator(BBa_K3033016).

Usage

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

As shown in Figure 2, except for the 11^th and 13^th bacterial colonies, other bacterial colonies did not grow under blue light. To further explore the lysis effect of pDawn(cI-LVA)-RBS070-LKD-T1-TOP10, we tracked the OD₆₀₀ values of pDawn(cI-LVA)-RBS070-LKD-T1-TOP10 and plotted the growth curves diagram. As shown in Figure 3, the lysis effect of pDawn(cI-LVA)-RBS070-LKD-T1-TOP10 meets our expectation in the first eight hours, but it is unstable after fifteen hours.

Figure 2: Growth conditions of E.coliTOP10-pDawn(cI-LVA)-RBS070-LKD-T1 in the dark and under the light.

Figure 3: Growth curves diagram of E.coli TOP10-pDawn(cI-LVA)-RBS070-LKD-T1.

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] [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).