Part:BBa_K4325027

pDawn-LKD-T1

Description

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

Usage

The pDawn (CI-LVA) blue light corresponding system (BBa_K1075044) and the lysis gene LKD (BBa_K4325004) were inserted into the pSEVA331 expression vector, which was inserted into E. coli TOP10 and screened out bacterial colony that grew in the dark but did not grow under blue light. Finally, the pSEVA331-pDawn (CI-LVA) -LKD-T1 plasmid was extracted and inserted into G. hansenii ATCC53582, after which we verified the responsiveness of pDawn (CI-LVA) to blue light.

Figure 1:

Gene circuit of pDawn (CI-LVA)-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 pDawn(CI-LVA)-LKD-T1-pSEVA331 in response to blue light lysis in E. coli TOP10.

As shown in Figure 2, except for the 16^th bacterial colony,almost all of the plaque did not grow under blue light To further show the lysis effect of pDawn (cI-LVA) -LKD-T1-pSEVA331-TOP10, we measured the OD ₆₀₀ values of pSEVA331-pDawn (cI-LVA) -LKD-T1-TOP10 periodically and ploting the growth curve diagram . As shown in Figure 3, pSEVA331-pDawn (cI-LVA) -LKD-T1-TOP10 was not excellent.

Figure 2:

Growth situation of pSEVA331-pDawn (cI-LVA) -LKD-T1-TOP10 in the dark and under the blue light.

Figure 3:

Growth curve diagram of pSEVA331-pDawn (cI-LVA) -LKD-T1-TOP10 in different growth conditions.

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

As shown in Figure 3, pDawn(cI-LVA)-LKD-T1 plasmid was successfully identified in G. hansenii ATCC53582 by agarose gel electrophoresis after electroporation the plasmid into our chassis bacteria.(electro-transforming)

Figure 4:Successfully identified by agarose gel electrophoresis of

(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.