Difference between revisions of "Part:BBa K4325018"
 
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<partinfo>BBa_K4325018 short</partinfo>
 
<partinfo>BBa_K4325018 short</partinfo>
 
===Description===
 
===Description===
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This composite part is a generator consisting  of pDawn(cI-LVA) (<partinfo>BBa_K1075044</partinfo>) and LKD-LVA (<partinfo>BBa_K4325011</partinfo>).
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This composite part is a generator consisting  of pDawn(cI-LVA)(<partinfo>BBa_K1075044</partinfo>),RBS070(<partinfo>BBa_K4325002</partinfo>), LKD-LVA (<partinfo>BBa_K4325011</partinfo>)and  T1 terminator(<partinfo>BBa_K3257021</partinfo>).
  
 
===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 with hydrolysis tag LKD-LVA (<partinfo>BBa_K4325011</partinfo>) into the pSEVA331 expression vector, which was inserted into <i> E. coli</i> TOP10 and screened for bacterial colony that grew in the dark but did not grow under blue light. Finally, the pDawn (cI-LVA)-LKD-LVA-T1 plasmids were inserted into <i>G. hansenii</i> ATCC53582 by electroporation to verify the responsiveness of pDawn(cI-LVA) to blue light.</p>
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<p>We inserted the pDawn(cI-LVA) blue light response system (<partinfo>BBa_K1075044</partinfo>) and the lysis gene with hydrolysis tag LKD-LVA (<partinfo>BBa_K4325011</partinfo>) into the pSEVA331 expression vector, which was incorporated into <i> E. coli</i> TOP10 and screened for bacterial colonies that grew in the dark but did not grow under blue light. Finally, the plasmids containing pDawn (cI-LVA)-LKD-LVA-T1 were electroporated into <i>G. hansenii</i> ATCC53582 to verify the responsiveness of pDawn(cI-LVA) to blue light.</p>
  
 
===Sequence and Features===
 
===Sequence and Features===
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=2022 SZPT-China=
 
=2022 SZPT-China=
 
<h3>Characterization</h3>
 
<h3>Characterization</h3>
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<h4>1.Batch screening of pDawn(cI-LVA)-LKD-LVA-T1 in response to blue light lysis in <i>E. coli</i>.</h4>  
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<h4>1.Batch screening of pDawn-RBS070-LKD-LVA-T1 by evaluating the response to blue light.</h4>  
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<p>As shown in Figure 1, We inserted 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 pDawn(cI-LVA)-RBS070-LKD-LVA-T1 was successfully expressed in <i>E. coli</i> TOP10.</p>
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<p>As shown in Figure 1, the plasmid containing pDawn(cI-LVA)-RBS070-LKD-LVA-T1 was introduced into <i>E. coli</i> TOP10 and performed the agarose gel electrophoresis. Then we performed the drop plate assay in the dark and under blue light. After 12 hours, we found that the 4<sup>th</sup>, 5<sup>th</sup>, 6<sup>th</sup>, 14<sup>th</sup> bacterial colonies grew in the dark and did not grow under blue light, (Figure 2) which indicates that pDawn(cI-LVA)-RBS070-LKD-LVA-T1 functions as intended in <i>.coli.</i> E</p>
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[[File:K18 1.png|600px|thumb|center|Figure 1:Successfully identified by agarose gel electrophoresis<p></p> 
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[[File:K18 1.png|600px|thumb|center|Figure 1: (1) pSEVA331-pDawn(cI)-X174E-T1; <b>(2) pSEVA331-pDawn(cI-LVA)-X174E-LVA-T1</b>; (3) pSEVA331-pDawn(cI)-LKD-T1; (4) pSEVA331-pDawn(cI-LVA)-RBS070-LKD-LVA-T1 were constructed successfully.]]
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<b>(1) pDawn (cI-LVA)-RBS070-LKD-LVA-T1</b> and (2) pDawn (cI-LVA)-X174E-LVA-T1 were successfully identified in <i>E. coli</i> TOP10]]
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[[File:K18 2.png|600px|thumb|center|Figure 2:Growth condition of pSEVA331-pDawn (cI-LVA)-RBS070-LKD-LVA-T1-TOP10 in the dark and under the light]]
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[[File:K18 2.png|600px|thumb|center|Figure 2:Growth condition of pDawn(cI-LVA)-RBS070-LKD-LVA-T1-TOP10 in the dark and under the light]]
  
  
  
 
<h3>References</h3>
 
<h3>References</h3>
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<p>[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.</p>
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<p> [1]Ohlendorf, R., Vidavski, R. R., Eldar, A., Moffat, K. & Möglich, A. From Dusk till Dawn: One-Plasmid Systems for Light-Regulated Gene Expression. J. Mol. Biol. <i>416</i>, 534-542 (2012).</p>
 +
<p> [2]Ceyssens, P.-J. et al. Genomic analysis of Pseudomonas aeruginosa phages LKD16 and LKA1: establishment of the phiKMV subgroup within the T7 supergroup. J. Bacteriol. <i>188</i>, 6924-31 (2006).</p>

Latest revision as of 14:41, 12 October 2022

pDawn-RBS070-LKD-LVA-T1

Description

This composite part is a generator consisting of pDawn(cI-LVA)(BBa_K1075044),RBS070(BBa_K4325002), LKD-LVA (BBa_K4325011)and T1 terminator(BBa_K3257021).

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 incorporated into E. coli TOP10 and screened for bacterial colonies that grew in the dark but did not grow under blue light. Finally, the plasmids containing pDawn (cI-LVA)-LKD-LVA-T1 were electroporated 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 pDawn-RBS070-LKD-LVA-T1 by evaluating the response to blue light.

As shown in Figure 1, the plasmid containing pDawn(cI-LVA)-RBS070-LKD-LVA-T1 was introduced into E. coli TOP10 and performed the agarose gel electrophoresis. Then we performed the drop plate assay in the dark and under blue light. After 12 hours, we found that the 4th, 5th, 6th, 14th bacterial colonies grew in the dark and did not grow under blue light, (Figure 2) which indicates that pDawn(cI-LVA)-RBS070-LKD-LVA-T1 functions as intended in .coli. E

Figure 1: (1) pSEVA331-pDawn(cI)-X174E-T1; (2) pSEVA331-pDawn(cI-LVA)-X174E-LVA-T1; (3) pSEVA331-pDawn(cI)-LKD-T1; (4) pSEVA331-pDawn(cI-LVA)-RBS070-LKD-LVA-T1 were constructed successfully.
Figure 2:Growth condition of pDawn(cI-LVA)-RBS070-LKD-LVA-T1-TOP10 in the dark and under the light


References

[1]Ohlendorf, R., Vidavski, R. R., Eldar, A., Moffat, K. & Möglich, A. From Dusk till Dawn: One-Plasmid Systems for Light-Regulated Gene Expression. J. Mol. Biol. 416, 534-542 (2012).

[2]Ceyssens, P.-J. et al. Genomic analysis of Pseudomonas aeruginosa phages LKD16 and LKA1: establishment of the phiKMV subgroup within the T7 supergroup. J. Bacteriol. 188, 6924-31 (2006).