Part:BBa_K3740032

Lysis Cassette LKD16

Description

The lysis cassette LKD16 is made of gp44 holin, gp45 endolysin, gp46 Rz-like protein and gp46.1 Rz1-like protein.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 252
  Illegal NgoMIV site found at 303
  
• 1000
  COMPATIBLE WITH RFC[1000]

2021 SZPT-China

Biology

Lysis Cassette LKD16 is originated from the genome of Pseudomonas aeruginosa bacteriophage LKD16, bacteriophage LKD16 was isolated from environmental water samples in Belgium. The lysis cassette LKD16 is made of gp44 holin (BBa_K3740013), gp45 endolysin (BBa_K3740014), gp46 Rz-like protein (BBa_K3740015) and gp46.1 Rz1-like protein (BBa_K3740016).

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.

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-LKD16-rrnB T1-pSEVA331-DH5α under blue light (a) and dark condition (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 condition (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.