Part:BBa_K4325020

pDawn-RBS070-X174E-T1

Description

This composite part is a generator consisting of pDawn(cI-LVA)(BBa_K1075044) and X174E(BBa_K1835500).

Usage

The pDawn (cI-LVA)(BBa_K1075044) blue light response system and the lysis gene X174E(BBa_K1835500) were inserted into the pSEVA331 expression vector,which wsa inserted into the E.coli TOP10,screened out bacterial colony that grew in the dark but did not grow under blue light. Finally, the pSEVA331-pDawn-RBS070-X174E-T1 plasmid was selected and inserted into G. hansenii ATCC53582 to verify the responsiveness of pDawn(cI-LVA) to blue light.

Figure 1:Gene circuit of pDawn(cI-LVA)-RBS070-X174E-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-X174E-T1 in response to blue light lysis in E. coli.

As shown in Figure 2, the 11^th and 15^th bacterial colony did not grow under blue light but grow in dark. In order to further explore the lysis effect of pSEVA331-pDawn (cI-LVA)-RBS070-X174E-T1-TOP10, we measured the OD₆₀₀ value of pSEVA331-pDawn (cI-LVA)-RBS070-X174E-T1-TOP10 periodically andploting the growth curve diagram.As shown in Figure 3, the lysis effect of pSEVA331-pDawn (cI-LVA)-RBS070-X174E-T1-TOP10 is not well.

Figure 2: The growth condition of pDawn(cI-LVA)-RBS070-X174E-T1-TOP10 in dark and under the blue light.

Figure 3: Growth curve diagram of pSEVA331-pDawn(cI-LVA)-RBS070-X174E-T1-TOP10.

2.pSEVA331-pDawn(cI-LVA)-RBS070-X174E-T1 in response to blue photolysis in G. hansenii ATCC53582.

pDawn(cI-LVA)-RBS070-X174E-T1 was inserted into G. hansenii ATCC53582 by electroporation and identified. As shown in Figure 4, pDawn(cI-LVA)-RBS070-X174E-T1 was successfully inserted into G. hansenii ATCC53582.

Figure4: Successfully identified by 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 and all of them were constructed successfully.

We transformed the pDawn (cI-LVA) -X174E-T1 plasmid into G. hansenii ATCC53582 and identified pDawn (cI-LVA) -X174E-T1 by using agarose gel electrophoresis. As shown in Figure 3, we successfully constructed the pDawn (cI-LVA) -X174E-T1.

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.