Difference between revisions of "Part:BBa K4325027"

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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K4325027 short</partinfo>
 
<partinfo>BBa_K4325027 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 (<partinfo>BBa_K4325004</partinfo>).
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This composite part is a generator consisting of  pDawn(cI-LVA) (<partinfo>BBa_K1075044</partinfo>), LKD (<partinfo>BBa_K4325004</partinfo>) and T1 terminator (<partinfo>BBa_K3033016</partinfo>).
 
===Usage===
 
===Usage===
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<p>The pDawn (CI-LVA) blue light corresponding system (<partinfo>BBa_K1075044</partinfo>) and the lysis gene LKD (<partinfo>BBa_K4325004</partinfo>) were inserted into the pSEVA331 expression vector, which was inserted into <i>E. coli</i> 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 <i>G. hansenii</i> ATCC53582, after which we verified the responsiveness of pDawn (CI-LVA) to blue light.</p>
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<p>The blue light responsive system pDawn(cI-LVA)(<partinfo>BBa_K1075044</partinfo>) and the lysis gene LKD (<partinfo>BBa_K4325004</partinfo>) were inserted 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 pDawn(cI-LVA)-LKD-T1 plasmid were extracted and electroporated into <i>G. hansenii</i> ATCC53582, after which we verified the responsiveness of pDawn(cI-LVA) to blue light.</p>
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[[File:K27 3.png|600px|thumb|center|Figure 1: <p></p>Gene circuit of pDawn (CI-LVA)-LKD-T1.]]
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[[File:K27 3.png|500px|thumb|center|Figure 1: Genetic circuit of pDawn(cI-LVA)-LKD-T1.]]
  
  
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<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K4325026 parameters</partinfo>
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<partinfo>BBa_K4325027 parameters</partinfo>
 
<!-- -->
 
<!-- -->
 
=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-T1-pSEVA331 in response to blue light lysis in <i>E. coli</i> TOP10.</h4>
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<h4>1.Batch screening of pDawn-LKD-T1 by evaluating the response to blue light.</h4>
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<p>As shown in Figure 2, except for the 16<sup>th</sup> 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 <sub>600</sub> 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.</p>
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<p>As shown in Figure 2, except for the 16<sup>th</sup> bacterial colony, almost all of the bacteria did not grow under blue light. To further show the lysis effect of pDawn(cI-LVA)-LKD-T1, we tracked the OD <sub>600</sub> values of pDawn(cI-LVA)-LKD-T1   and plotted the growth curves diagram. As shown in Figure 3, <i>E. coli</i> TOP10-pSEVA331-pDawn(cI-LVA)-LKD-T1 is not excellent.</p>
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[[File:K27 2.png|600px|thumb|center|Figure 2:Growth situation of pSEVA331-pDawn (cI-LVA) -LKD-T1-TOP10 in the dark and under the blue light.]]
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[[File:K27 2.png|400px|thumb|center|Figure 2: Growth situation of <i>E. coli</i> TOP10-pSEVA331-pDawn(cI-LVA)-LKD-T1 in the dark and under blue light.]]
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[[File:K27 4.png|600px|thumb|center|Figure 3:Growth curve diagram of pSEVA331-pDawn (cI-LVA) -LKD-T1-TOP10 in different growth conditions.]]
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[[File:K27 1.png|400px|thumb|center|Figure 3: Growth curves diagram of <i>E. coli</i> TOP10-pSEVA331-pDawn(cI-LVA)-LKD-T1 in different growth conditions.]]
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<h3>2.pSEVA331-pDawn(cI-LVA)-LKD-T1 in response to blue photolysis in <i>G. hansenii</i> ATCC53582.</h3>
 
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<p>As shown in Figure 3, pDawn(cI-LVA)-LKD-T1 plasmid was successfully identified in <i>G. hansenii</i> ATCC53582 by agarose gel electrophoresis after transformed the plasmid into our chassis bacteria.</p>
 
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[[File:K27 5.png|600px|thumb|center|Figure 4:Successfully identified by agarose gel electrophoresis of  <p></p>
 
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(1)pSEVA331-pDawn(cI-LVA)-RBS070-LKD-T1-<i>G. hansenii</i> ATCC53582 <p></p>
 
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(2)pSEVA331-pDawn (cI-LVA)-LKD-T1-<i>G. hansenii</i> ATCC53582 <p></p>
 
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(3)pSEVA331-pDawn(cI-LVA)-RBS070-X174E-T1-<i>G. hansenii</i> ATCC53582 <p></p>
 
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(4)pSEVA331-pDawn(CI-LVA)-X174E-T1-<i>G. hansenii</i> ATCC53582]]
 
 
<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] 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>[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).</p>

Latest revision as of 14:38, 12 October 2022


pDawn-LKD-T1

Description

This composite part is a generator consisting of pDawn(cI-LVA) (BBa_K1075044), LKD (BBa_K4325004) and T1 terminator (BBa_K3033016).

Usage

The blue light responsive system pDawn(cI-LVA)(BBa_K1075044) and the lysis gene LKD (BBa_K4325004) were inserted 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 pDawn(cI-LVA)-LKD-T1 plasmid were extracted and electroporated into G. hansenii ATCC53582, after which we verified the responsiveness of pDawn(cI-LVA) to blue light.

Figure 1: Genetic 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-LKD-T1 by evaluating the response to blue light.

As shown in Figure 2, except for the 16th bacterial colony, almost all of the bacteria did not grow under blue light. To further show the lysis effect of pDawn(cI-LVA)-LKD-T1, we tracked the OD 600 values of pDawn(cI-LVA)-LKD-T1 and plotted the growth curves diagram. As shown in Figure 3, E. coli TOP10-pSEVA331-pDawn(cI-LVA)-LKD-T1 is not excellent.

Figure 2: Growth situation of E. coli TOP10-pSEVA331-pDawn(cI-LVA)-LKD-T1 in the dark and under blue light.
Figure 3: Growth curves diagram of E. coli TOP10-pSEVA331-pDawn(cI-LVA)-LKD-T1 in different growth conditions.


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.

[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).