Part:BBa_K4325026

pDawn-RBS070-LKD-T1

Description

This composite part is a generator containing pDawn (CI-LVA) (BBa_K1075044) and LKD (BBa_K4325004).

Usage

The pDawn (CI-LVA) blue light responsive system (BBa_K1075044) and lysis cassette LKD (BBa_K4325004) were linked with the pSEVA331 expression vector. Then these constructs were transformed into . E. coli TOP10 and screened for strain that grew well in dark but failed to grow under blue light. Finally, the pSEVA331-pDawn-RBS070-LKD-T1 plasmid were obtained and genomically inserted into G. hansenii ATCC53582 for further responsiveness verification 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.Characterization in E. coli TOP10

As shown in Figure1, 14 colonies meet our expectation and one of them was picked and culture in the dark and under blue light. Growth curves shows the bacteria continue growing after 8 hours, indicating this system is not robust.

Figure 2:

(a) Colony growth of E. coli TOP10 containing pSEVA331-pDawn(cI-LVA)-RBS070-LKD-T1 in the dark

(a2) Colony growth of E. coli TOP10 containing pSEVA331-pDawn(cI-LVA)-RBS070-LKD-T1 under blue light

(a3) Colony growth of E. coli TOP10-pSEVA331-pDawn(cI-LVA)-RBS070-LKD-T1;

2.Characterization in G. hansenii ATCC53582

The picked plasmids from E. coli TOP10 were transformed into G. hansenii ATCC53582. It shows that these two plasmids still maintain their excellent lysis effect in G. hansenii ATCC53582.

Figure 3:

(a1) Colony growth of G. hansenii ATCC53582 containing pSEVA331-pDawn(cI-LVA)-RBS070-LKD-T1 in the dark

(a2) Colony growth of G. hansenii ATCC53582 containing pSEVA331-pDawn(cI-LVA)-RBS070-LKD-T1 under blue light

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.