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 response system (BBa_K1075044) and lysis gene LKD (BBa_K4325004) were inserted into the pSEVA331 expression vector, which was inserted into E. coli TOP10, screened out the bacterial colony which grew in the dark but did not grow under blue light. Finally, the pSEVA331-pDawn-RBS070-LKD-T1 plasmid was selected and inserted into G. hansenii ATCC53582 by electroporation to verify the responsiveness of pDawn (cI-LVA) under 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.Batch screening of pSEVA331-pDawn(cI-LVA)-RBS070-LKD-T1 in response to blue light lysis in E. coli TOP10.

As shown in Figure 2,except for the 11^th and 13^th bacterial colony,almost all of the bacterial colony did not grow under blue light. To further explore the lysis effect of pSEVA331-pDawn (cI-LVA) -RBS070-LKD-T1-TOP10, we measured the OD ₆₀₀ values of pSEVA331-pDawn (cI-LVA) -RBS070-LKD-T1-TOP10 and ploting the growth curve diagram. As shown in Figure 3, the pSEVA331-pDawn (cI-LVA) -RBS070-LKD-T1-TOP10 lysis effect was well in the first eight hours, but was unstable after fifteen hours.

Figure 2: Growth condition of pDawn (cI-LVA)-RBS070-LKD-T1-TOP10 in the dark and under the light

Figure 3: Growth curve diagramof pDawn (cI-LVA)-RBS070-LKD-T1-TOP10

2.pSEVA331-pDawn(cI-LVA)-RBS070-LKD-T1 in response to blue photolysis in G. hansenii ATCC53582.

As shown in Figure 3, we successfully indicated the pDawn (cI-LVA) -RBS070-LKD-T1 into G. hansenii ATCC53582 by electroporation

Figure 4: Successfully identification with agarose gel electrophoresis

(1)pSEVA331-pDawn(cI-LVA)-RBS070-LKD-T1-G. hansenii ATCC53582

(2)pSEVA331-pDawn (cI-LVA)-LKD-T1-G. hansenii ATCC53582

(3)pSEVA331-pDawn(cI-LVA)- RBS070-X174E-T1-G. hansenii ATCC53582

(4)pSEVA331-pDawn(cI-LVA)-X174E-T1-G. hansenii ATCC53582

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.