Difference between revisions of "Part:BBa K4325018"

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(→‎2022 SZPT-China)
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<h3>Characterization</h3>
 
<h3>Characterization</h3>
 
<h4>1.Batch screening of pSEVA331-pDawn(cI-LVA)-LKD-LVA-T1 in response to blue light lysis in <i>E. coli</i>.</h4>  
 
<h4>1.Batch screening of pSEVA331-pDawn(cI-LVA)-LKD-LVA-T1 in response to blue light lysis in <i>E. coli</i>.</h4>  
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<p>As shown in Figure 1, We inserted pSEVA331-pDawn (cI-LVA) -RBS070-LKD-LVA-T1 into <i>E. coli</i> TOP10 and performed the agarose gel electrophoresis. Then we cultured monocloning site in the dark and under the light. After 12 hours, we found that the 4<sup>th</sup>, 5<sup>th</sup>, 6<sup>th</sup>, 14<sup>th</sup> bacteria grew in the dark and did not grow under the light,(Figure 2) which indicates that pSEVA331-pDawn (cI-LVA) -RBS070-LKD-LVA-T1 is successfully expressed in <i>E. coli</i> TOP10.</p>
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<p>As shown in Figure 1, We inserted pSEVA331-pDawn (cI-LVA) -RBS070-LKD-LVA-T1 into <i>E. coli</i> TOP10 and performed the agarose gel electrophoresis. Then we cultured monocloning site in the dark and under the light. After 12 hours, we found that the 4<sup>th</sup>, 5<sup>th</sup>, 6<sup>th</sup>, 14<sup>th</sup> bacteria grew in the dark and did not grow under the light,(Figure 2) which indicates that pSEVA331-pDawn (cI-LVA) -RBS070-LKD-LVA-T1 was successfully expressed in <i>E. coli</i> TOP10.</p>
 
[[File:K18 1.png|600px|thumb|center|Figure 1:Successfully identified by agarose gel electrophoresis of  <p></p>  
 
[[File:K18 1.png|600px|thumb|center|Figure 1:Successfully identified by agarose gel electrophoresis of  <p></p>  
 
(1)pSEVA331-pDawn (cI-LVA)-RBS070-LKD-LVA-T1 and
 
(1)pSEVA331-pDawn (cI-LVA)-RBS070-LKD-LVA-T1 and

Revision as of 13:01, 10 October 2022

pDawn-RBS070-LKD-LVA-T1

Description

This composite part is a generator consisting of pDawn (cI-LVA) (BBa_K1075044) and LKD-LVA (BBa_K4325011).

Usage

We inserted the pDawn (cI-LVA) blue light response system (BBa_K1075044) and the lysis gene with hydrolysis tag LKD-LVA (BBa_K4325011) into the pSEVA331 expression vector, which was inserted into E. coli TOP10 and screened for bacterial colony that grew in the dark but did not grow under blue light. Finally, the pSEVA331-pDawn (cI-LVA) -LKD-LVA-T1 plasmids were inserted into G. hansenii ATCC53582 by electroporation 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)-LKD-LVA-T1 in response to blue light lysis in E. coli.

As shown in Figure 1, We inserted pSEVA331-pDawn (cI-LVA) -RBS070-LKD-LVA-T1 into E. coli TOP10 and performed the agarose gel electrophoresis. Then we cultured monocloning site in the dark and under the light. After 12 hours, we found that the 4th, 5th, 6th, 14th bacteria grew in the dark and did not grow under the light,(Figure 2) which indicates that pSEVA331-pDawn (cI-LVA) -RBS070-LKD-LVA-T1 was successfully expressed in E. coli TOP10.

Figure 1:Successfully identified by agarose gel electrophoresis of

(1)pSEVA331-pDawn (cI-LVA)-RBS070-LKD-LVA-T1 and (2)pSEVA331-pDawn (cI-LVA)-X174E-LVA-T1 were successfully identified in E. coli TOP10
Figure 2:Growth situation of pSEVA331-pDawn (cI-LVA)-RBS070-LKD-LVA-T1-TOP10 in the dark and under the light


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.