Part:BBa_K4325024

pDawn(cl)-X174E-T1

Description

This composite part is a generator consisting of pDawn (without LVA tag) (BBa_K4325010) and X174E (BBa_K1835500).

Usage

We inserted the pDawn (without LVA tag) (BBa_K4325010)blue light response system (BBa_K4325010)with the lysis gene X174E (BBa_K1835500)into the pSEVA331 expression vector, which was inserted into E. coli TOP10 . The bacterial colony that grew in the dark but did not grow under blue light were screened to verify the responsiveness of pDawn (without LVA tag) to blue light.

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 pDawn(without LVA tag)-X174E-T1 in response to blue light lysis in E. coli.

Figure1. (1)pDawn(CI-LVA)-X174E-LVA-T1-pSEVA331; (2)pDawn(CI)-X174E-T1-pSEVA331; (3)pDawn(CI-LVA)-RBS070-LKD-LVA-T1-pSEVA331; (4)pDawn(CI)-RBS070-LKD-T1-pSEVA331 were constructed successfully.

<P>

Figure2. Growth condition of pDawn(CI)-X174E-T1-pSEVA331-TOP10 in dark and under light.

As shown in Figure 1, we successfully constructed pDawn(CI)-X174E-T1-pSEVA331 and transformed it into E.coli TOP10, selection a monoclonal colony and culturing in different growth conditions. As shown in Figure 2, the 9th colony of pDawn (CI)-X174E-T1-pSEVA331-TOP10 grew in the dark, but did not grow under blue light.

References

Robert Ohlendorf 1 , Roee R. Vidavski 2 , Avigdor Eldar 2, Keith Moffat 3,4 and Andreas Möglich 1, 3.From Dusk till Dawn: One-Plasmid Systems for Light-Regulated Gene Expression.