Part:BBa_K3740044

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 in 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 2, the 4^th 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.

Figure 2：The growth status of pDawn-RBSNNN-X174 E-rrnB T1-pSEVA331-DH5α under blue light (a) and dark conditions (b).

2. pDawn-RBSNNN-X174 E-rrnB T1-pSEVA331-7# in response to blue phptolysis in G. hansenii ATCC 53582

Method: We launched the liquid culture of pDawn-RBSNNN-X174 E-rrnB T1-pSEVA331-DH5α, extracted the plasmid and introduced into G. hansenii ATCC 53582. After 2 days of incubation, we selected 20 monoclonal to spot on two parallel new plates, which were incubated for another 2 days, with one under blue light and the other in the dark. The strains with obvious growth difference under different illumination conditions were preserved. The drop plate assay using this strain was repeated to verify the lysis effect of X174 E on G. hansenii ATCC 53582.

As shown in Figure 3 (a), G. hansenii ATCC 53582 strains under the dark condition exhibited better growth than those under blue light irradiation; (b) pDawn-RBSNNN-X174 E-rrnB T1-pSEVA331-G. hansenii ATCC 53582-7# showed a stable lysis effect under blue light illumination but not in the dark, indicating that we successfully expressed the cleavage protein X174E in G. hansenii ATCC 53582.

Figure 3. (a) The growth status of pDawn-RBSNNN-X174 E-rrnB T1-pSEVA331-G. hansenii ATCC 53582-4# under blue light (1) and dark condition (2). (b)The growth status of pDawn-RBSNNN-X174 E-rrnB T1-pSEVA331-G. hansenii ATCC 53582-7# under blue light (1) and dark condition (2).

References

[1] Witte A, Wanner G, Sulzner M, et al. Dynamics of PhiX174 protein E-mediated lysis of Escherichia coli[J]. Archives of Microbiology, 1992, 157(4):381-388.

Phi X 147 E Lysis Gene: BBa_K1835500

2022 SZPT-China

Characterization

1.Batch screening of pSEVA331-pDawn-RBSNNN-X174 E-rrnB T1 in response to blue light lysis in E. coli TOP10.

We performed a new round of phenotyping assay for pDawn-RBSNNN-X174E-rrnB T1. First, the monoclonal transformants of E. coli TOP10 with pDawn-RBSNNN-X174E-rrnB T1 was picked and grown on two parallel new LB plates, with one cultured in the dark and the other under blue light respectively to screen for correct builds. As shown in Figure 1,pSEVA331-pDawn-RBSNNN-X174E-rrnB T1 had a total of 10 strains respectively in which the genetic construct works as intended. This suggests that the lysis protein X174E can be successfully expressed in the E. coli TOP10 under our designed illumination conditions.

Figure 1:

(a) Colony growth of E. coli TOP10 containing pDawn-RBSNNN-X174E-rrnB T1 in the dark.

(b) Colony growth of E. coli TOP10 containing pDawn-RBSNNN-X174E-rrnB T1 under blue light.

From the above successul recombinant candidates(red squares in Figure 1), we selected bacterial strains that can gwplyse under blue light, launched two copies of bacterial liquid culture with agitation under either light or dark condition and measured the OD ₆₀₀ values. As shown in Figure 2, we found that the lysis protein can accumulate with time, and the bacteria began to be lysed at 3 or 4 hours post inoculation, since the OD₆₀₀ value decreased, suggesting that the lysis protein reaches the lethal concentration at this time point..

Figure 2:

(a) Growth curve of E. coli TOP10 containing pSEVA331-pDawn-RBSNNN-X174E-rrnB T1 #1

(b) Growth curve of E. coli TOP10 containing pSEVA331-pDawn-RBSNNN-X174E-rrnB T1 #11