Part:BBa_K4325021

pDawn-X174E-T1

Description

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

Usage

We inserted the pDawn (CI-LVA) blue light response system (BBa_K1075044)and the lysis gene X174E (BBa_K1835500) into the pSEVA331 expression vector, which was inserted into E. coli TOP10 and screened for colonies that grew in the dark but did not grow under blue light. Finally, the pSEVA331-pDawn (CI-LVA) -X174E-T1 plasmids were selected and further inserted into G. hansenii ATCC53582 to verify the responsiveness of pDawn (CI-LVA) to blue light.

[[File:PDawn.png |600px|thumb|center|Figure 1:Gene circuit of pDawn(cI-LVA)-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)-X174E-T1 in response to blue light lysis in E. coli.

[[File:PDawn1.png |600px|thumb|center|Figure 2: Growth condition of pSEVA331-pDawn (CI-LVA) -LKD-T1-TOP10 in the dark and under light.]]

[[File:PDawn2.png |600px|thumb|center|Figure 3: Growth curve diagram of pSEVA331-pDawn (CI-LVA) -X174E-T1-pSEVA331-TOP10.]]

As shown in Figure 2, the colony numbers 1st, 4th, 5th, 6th, 7th, 8th, 10th, 14th, and 16th met our expectation. To further searching the lysis effect of pDawn (CI-LVA) -X174E-T1-pSEVA331-TOP10, we measured the OD600 value of pDawn (CI-LVA) -X174E-T1-pSEVA331-TOP10 periodically and ploting the growth curve diagram. As shown in Figure 3, the lysis effect of pDawn (CI-LVA) -X174E-T1-pSEVA331-TOP10 is mediocre.

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

Figure3: 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

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.