Difference between revisions of "Part:BBa K4325021"
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[[File:pDawn.png|600px|thumb|center|Figure 1: Gene circuit of pDawn(CI-LVA)-X174E-T1.]] <p></p>
 
[[File:pDawn.png|600px|thumb|center|Figure 1: Gene circuit of pDawn(CI-LVA)-X174E-T1.]] <p></p>
 
===Usage===
 
===Usage===
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<p>We inserted the pDawn (cI-LVA) blue light response system (<partinfo>BBa_K1075044</partinfo>)and the lysis gene X174E (<partinfo>BBa_K1835500</partinfo>) into the pSEVA331 expression vector, which was inserted into <i>E. coli</i> 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 <i>G. hansenii</i>  ATCC53582 to verify the responsiveness of pDawn (CI-LVA) to blue light.
+
<p>We inserted the pDawn (cI-LVA) blue light response system (<partinfo>BBa_K1075044</partinfo>)and the lysis gene X174E (<partinfo>BBa_K1835500</partinfo>) into the pSEVA331 expression vector, which was inserted into <i>E. coli</i> TOP10 and screened out 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 <i>G. hansenii</i>  ATCC53582 to verify the responsiveness of pDawn (CI-LVA) to blue light.
 
</p>
 
</p>
  

Revision as of 06:35, 11 October 2022

pDawn-X174E-T1

Description

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

Figure 1: Gene circuit of pDawn(CI-LVA)-X174E-T1.

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 out 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.

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.

Figure 2: Growth condition of pSEVA331-pDawn (cI-LVA) -LKD-T1-TOP10 in the dark and under light.

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 pSEVA331-pDawn (cI-LVA) -X174E-T1-TOP10 periodically and ploting the growth curve diagram. As shown in Figure 3, the lysis effect of pSEVA331-pDawn (cI-LVA) -X174E-T1-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.