Part:BBa_K3740033

pDawn-B0034-LKD16-rrnB T1

Description

This composite part is a generator consisting of the pDawn promoter and LKD16.

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 LKD16 (BBa_K3740032).

Usage

The target gene of pDawn blue light response system (BBa_K1075044) and Lysis Cassette LKD16 (BBa_K3740032) were insert into the pSEVA331 expression vector. Then, random primer guided mutagenesis method to modulate the strength of the RBS (BBa_B0034) located upstream of LKD16. After introducing into E. coli DH5α, E. coli isolates that normally grow in the dark and cannot under the light of 470nm blue light were screened. Finally, the pDawn-RBSNNN-LKD16-rrnB T1 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 LKD16(BBa_K3740032).

Characterization

1.Batch screening of pDawn-RBSNNN-LKD16-rrnB T1-pSEVA331 in response to blue light lysis in E. coli.

As shown in Figure 2, the 4^th, 9^th, 10^th and 12^th of the plate can grow normally in the dark, but not grow under blue light, indicating that LKD16 can induce lysis in E. coli in response to blue light.

Figure 2. The growth status of pDawn-RBSNNN-rrnB T1-LKD16-pSEVA331-DH5α under blue light (a) and dark conditions (b).

2. pDawn-RBSNNN-LKD16-rrnB T1-pSEVA331-8# in response to blue photolysis in G. hansenii ATCC 53582.

As shown in Figure 3, although 7^th, 8^th, 9^th, 13^th and 17^th on the plate can grow under the dark, not grow under blue light, the growth of the bacteria under dark conditions is abnormal. Therefore, although the LKD16 cleavage protein has a cleavage function, its function is not very stable.

Figure 3. pDawn-RBSNNN-LKD16-rrnB T1-pSEVA331-G.hansenii ATCC 53582-8# cultured under blue light (a) and dark conditions (b).

References

[1] Ceyssens P J, Lavigne R, Mattheus W, et al. Genomic Analysis of Pseudomonas aeruginosa Phages LKD16 and LKA1: Establishment of the KMV Subgroup within the T7 Supergroup[J]. Journal of Bacteriology, 2006.