Difference between revisions of "Part:BBa K4325026"
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K4325026 short</partinfo>
 
<partinfo>BBa_K4325026 short</partinfo>
 
===Description===
 
===Description===
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This composite part is a generator containing pDawn (CI-LVA) (<partinfo>BBa_K1075044</partinfo>) and LKD (<partinfo>BBa_K4325004</partinfo>).
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This composite part is a generator containing pDawn (cI-LVA) (<partinfo>BBa_K1075044</partinfo>) and LKD (<partinfo>BBa_K4325004</partinfo>).
 
===Usage===
 
===Usage===
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<p>The pDawn (CI-LVA) blue light responsive system (<partinfo>BBa_K1075044</partinfo>) and lysis cassette LKD (<partinfo>BBa_K4325004</partinfo>) were linked with the pSEVA331 expression vector. Then these constructs were transformed into <i>. E. coli</i> TOP10 and screened for strain that grew well in dark but failed to grow under blue light. Finally, the pSEVA331-pDawn-RBS070-LKD-T1 plasmid were obtained and genomically inserted into <i>G. hansenii </i> ATCC53582 for further responsiveness verification of pDawn (CI-LVA) to blue light.</p>
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<p>The pDawn (cI-LVA) blue light response system (<partinfo>BBa_K1075044</partinfo>) and lysis gene LKD (<partinfo>BBa_K4325004</partinfo>) were inserted into the pSEVA331 expression vector, which was inserted into <i>E. coli</i> TOP10, screen out the bacterial colony that grew in the dark but did not grow under blue light Finally, the pSEVA331-pDawn-RBS070-LKD-T1 plasmid was extracted and inserted by electroporation into <i> G. hansenii</i> ATCC53582 to verify the responsiveness of pDawn (cI-LVA) to blue light.</p>
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[[File:K26 3.png|600px|thumb|center|Figure 1: <p></p>Gene circuit of pDawn (CI-LVA)-RBS070-LKD-T1.]]
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[[File:K26 1.png|600px|thumb|center|Figure 1: <p></p>Gene circuit of pDawn (cI-LVA)-RBS070-LKD-T1.]]
  
  
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=2022 SZPT-China=
 
=2022 SZPT-China=
 
<h3>Characterization</h3>
 
<h3>Characterization</h3>
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<h4>1.Characterization in <i>E. coli</i> TOP10</h4>
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<h4>1.Batch screening of pSEVA331-pDawn(cI-LVA)-RBS070-LKD-T1 in response to blue light lysis in <i>E. coli</i> TOP10.</h4>
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<p>As shown in Figure1, 14 colonies meet our expectation and one of them was picked and culture in the dark and under blue light. Growth curves shows the bacteria continue growing after 8 hours, indicating this system is not robust.</p>
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[[File:K26 1.png|600px|thumb|center|Figure 2: <p></p>(a) Colony growth of <i>E. coli</i> TOP10 containing pSEVA331-pDawn(cI-LVA)-RBS070-LKD-T1 in the dark <p></p>
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<p>As shown in Figure 2,except for the 11th and 13<sup>th </sup> bacterial colony,almost all of the bacterial colony did not grow under blue light.To further explore the lysis effect of pSEVA331-pDawn (cI-LVA) -RBS070-LKD-T1-TOP10, we measured the OD <sub>600</sub> values of pSEVA331-pDawn (cI-LVA) -RBS070-LKD-T1-TOP10 and ploting the growth curve diagram.As shown in Figure 3, the pSEVA331-pDawn (cI-LVA) -RBS070-LKD-T1-TOP10 lysis effect was well in the first eight hours, but was unstable after fifteen hours.</p>
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(a2) Colony growth of <i>E. coli</i> TOP10 containing pSEVA331-pDawn(cI-LVA)-RBS070-LKD-T1 under blue light <p></p>
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(a3) Colony growth of <i>E. coli</i> TOP10-pSEVA331-pDawn(cI-LVA)-RBS070-LKD-T1;]]
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[[File:K26 2.png|600px|thumb|center|Figure 2: <p></p>Growth condition of pDawn (cI-LVA)-RBS070-LKD-T1-TOP10 in the dark and under the light]]
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<h4>2.Characterization in <i>G. hansenii</i> ATCC53582</h4>
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[[File:K26 3.png|600px|thumb|center|Figure 3: <p></p> Growth curve diagramof pDawn (cI-LVA)-RBS070-LKD-T1-TOP10]]
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<p>The picked plasmids from <i> E. coli</i> TOP10 were transformed into <i> G. hansenii</i> ATCC53582. It shows that these two plasmids still maintain their excellent lysis effect in <i>G. hansenii</i> ATCC53582.</p>
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[[File:K26 2.png|600px|thumb|center|Figure 3: <p></p>(a1) Colony growth of <i>G. hansenii</i> ATCC53582 containing pSEVA331-pDawn(cI-LVA)-RBS070-LKD-T1 in the dark <p></p>
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(a2) Colony growth of <i>G. hansenii</i> ATCC53582 containing pSEVA331-pDawn(cI-LVA)-RBS070-LKD-T1 under blue light]]
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<h4>pSEVA331-pDawn(cI-LVA)-RBS070-LKD-T1 in response to blue photolysis in <i>G. hansenii</i> ATCC53582.</h4>
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<p>As shown in Figure 3, we successfully indicated the  pDawn (cI-LVA) -RBS070-LKD-T1 into <i>G. hansenii</i> ATCC53582 by electroporation</p>
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 +
[[File:K26 4.png|600px|thumb|center|Figure 4: <p></p>Successfully identification withagarose gel electrophoresis <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 ]]
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<h3>References</h3>
 
<h3>References</h3>
 
<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>
 
<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>

Revision as of 10:17, 10 October 2022

pDawn-RBS070-LKD-T1

Description

This composite part is a generator containing pDawn (cI-LVA) (BBa_K1075044) and LKD (BBa_K4325004).

Usage

The pDawn (cI-LVA) blue light response system (BBa_K1075044) and lysis gene LKD (BBa_K4325004) were inserted into the pSEVA331 expression vector, which was inserted into E. coli TOP10, screen out the bacterial colony that grew in the dark but did not grow under blue light Finally, the pSEVA331-pDawn-RBS070-LKD-T1 plasmid was extracted and inserted by electroporation into G. hansenii ATCC53582 to verify the responsiveness of pDawn (cI-LVA) to blue light.

Figure 1:

Gene circuit of pDawn (cI-LVA)-RBS070-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 pSEVA331-pDawn(cI-LVA)-RBS070-LKD-T1 in response to blue light lysis in E. coli TOP10.

As shown in Figure 2,except for the 11th and 13th bacterial colony,almost all of the bacterial colony did not grow under blue light.To further explore the lysis effect of pSEVA331-pDawn (cI-LVA) -RBS070-LKD-T1-TOP10, we measured the OD 600 values of pSEVA331-pDawn (cI-LVA) -RBS070-LKD-T1-TOP10 and ploting the growth curve diagram.As shown in Figure 3, the pSEVA331-pDawn (cI-LVA) -RBS070-LKD-T1-TOP10 lysis effect was well in the first eight hours, but was unstable after fifteen hours.

Figure 2:

Growth condition of pDawn (cI-LVA)-RBS070-LKD-T1-TOP10 in the dark and under the light
Figure 3:

Growth curve diagramof pDawn (cI-LVA)-RBS070-LKD-T1-TOP10


pSEVA331-pDawn(cI-LVA)-RBS070-LKD-T1 in response to blue photolysis in G. hansenii ATCC53582.

As shown in Figure 3, we successfully indicated the pDawn (cI-LVA) -RBS070-LKD-T1 into G. hansenii ATCC53582 by electroporation

File:K26 4.png
Figure 4:

Successfully identification withagarose 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

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.