pDawn-LKD-T1

Description

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

Usage

The blue light responsive system pDawn(cI-LVA)(BBa_K1075044) and the lysis gene LKD (BBa_K4325004) were inserted into the pSEVA331 expression vector, which was incorporated into pSEVA331-E. coli TOP10 and screened for bacterial colonies that grew in the dark but did not grow under blue light. Finally, the pDawn(cI-LVA)-LKD-T1 plasmid were extracted and electroporated into G. hansenii ATCC53582, after which we verified the responsiveness of pDawn(cI-LVA) to blue light.

Figure 1: Genetic circuit of pDawn(cI-LVA)-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)-LKD-T1 in response to blue light lysis in E. coli TOP10.

As shown in Figure 2, except for the 16^th bacterial colony, almost all of the bacteria did not grow under blue light. To further show the lysis effect of pDawn(cI-LVA)-LKD-T1, we tracked the OD ₆₀₀ values of pDawn(cI-LVA)-LKD-T1 and plotted the growth curves diagram. As shown in Figure 3,E. coli TOP10-pSEVA331-pDawn(cI-LVA)-LKD-T1 was not excellent.

Figure 2: Growth situation of E. coli TOP10-pSEVA331-pDawn(cI-LVA)-LKD-T1 in the dark and under blue light.

Figure 3: Growth curves diagram of E. coli TOP10-pSEVA331-pDawn(cI-LVA)-LKD-T1 in different growth conditions.

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] 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).