Difference between revisions of "Part:BBa K4325026"
 
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<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) and LKD.
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This composite part is a generator containing pDawn(cI-LVA) (<partinfo>BBa_K1075044</partinfo>), RBS070 (<partinfo>BBa_K4325002</partinfo>), LKD (<partinfo>BBa_K4325004</partinfo>) and  T1 terminator(<partinfo>BBa_K3033016</partinfo>).
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===Biology===
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===Usage===
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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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<p>We inserted the blue light responsive system pDawn(cI-LVA) (BBa_K1075044) and lysis gene LKD (BBa_K4325004) into the pSEVA331 expression vector, which was incorporated into <i>E. coli</i> TOP10 and screened for the bacterial colonies which grew in the dark but did not grow under blue light. Finally, the plasmids containing pDawn(cI-LVA)-RBS070-LKD-T1 were selected and introduced into <i>G. hansenii</i> ATCC53582 by electroporation to verify the responsiveness of pDawn(cI-LVA) to blue light.</p>
  
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[[File:K26 11.png|600px|thumb|center|Figure 1: Gene circuit of pDawn(cI-LVA)-RBS070-LKD-T1.]]
  
 
===Sequence and Features===
 
===Sequence and Features===
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<partinfo>BBa_K4325026 parameters</partinfo>
 
<partinfo>BBa_K4325026 parameters</partinfo>
 
<!-- -->
 
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===2022 SZPT-China=
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=2022 SZPT-China=
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<h3>Biology</h3>
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<p>This composite part is a generator containing pDawn (CI-LVA) (<partinfo>BBa_K1075044</partinfo>) and LKD (<partinfo>BBa_K4325004</partinfo>).</p>
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<h3>Characterization</h3>
 
<h3>Characterization</h3>
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<h4>Characterization in <i>E. coli</i>  TOP10</h4>
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<h4>1.Batch screening of pDawn-RBS070-LKD-T1 by evaluating the response to blue light.</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 1: <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 11<sup>th</sup> and 13<sup>th</sup> bacterial colonies, other bacterial colonies did not grow under blue light. To further explore the lysis effect of pDawn(cI-LVA)-RBS070-LKD-T1-TOP10, we tracked the OD<sub>600</sub> values of pDawn(cI-LVA)-RBS070-LKD-T1-TOP10 and plotted the growth curves diagram. As shown in Figure 3, the lysis effect of pDawn(cI-LVA)-RBS070-LKD-T1-TOP10 meets our expectation in the first eight hours, but it is 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: Growth conditions of <i>E.coli</i>TOP10-pDawn(cI-LVA)-RBS070-LKD-T1 in the dark and under the light.]]
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<h4>Characterization in <i>G. hansenii</i> ATCC53582</h4>
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[[File:K26 33.png|600px|thumb|center|Figure 3: Growth curves diagram of <i>E.coli</i> TOP10-pDawn(cI-LVA)-RBS070-LKD-T1.]]
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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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<h3>References</h3>
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[[File:K26 2.png|600px|thumb|center|Figure 2: <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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<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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(a2) Colony growth of <i>G. hansenii</i> ATCC53582 containing pSEVA331-pDawn(cI-LVA)-RBS070-LKD-T1 under blue light]]
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<p>[2] [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 14:40, 12 October 2022

pDawn-RBS070-LKD-T1

Description

This composite part is a generator containing pDawn(cI-LVA) (BBa_K1075044), RBS070 (BBa_K4325002), LKD (BBa_K4325004) and T1 terminator(BBa_K3033016).

Usage

We inserted the blue light responsive system pDawn(cI-LVA) (BBa_K1075044) and lysis gene LKD (BBa_K4325004) into the pSEVA331 expression vector, which was incorporated into E. coli TOP10 and screened for the bacterial colonies which grew in the dark but did not grow under blue light. Finally, the plasmids containing pDawn(cI-LVA)-RBS070-LKD-T1 were selected and introduced into G. hansenii ATCC53582 by electroporation 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 pDawn-RBS070-LKD-T1 by evaluating the response to blue light.

As shown in Figure 2, except for the 11th and 13th bacterial colonies, other bacterial colonies did not grow under blue light. To further explore the lysis effect of pDawn(cI-LVA)-RBS070-LKD-T1-TOP10, we tracked the OD600 values of pDawn(cI-LVA)-RBS070-LKD-T1-TOP10 and plotted the growth curves diagram. As shown in Figure 3, the lysis effect of pDawn(cI-LVA)-RBS070-LKD-T1-TOP10 meets our expectation in the first eight hours, but it is unstable after fifteen hours.

Figure 2: Growth conditions of E.coliTOP10-pDawn(cI-LVA)-RBS070-LKD-T1 in the dark and under the light.
Figure 3: Growth curves diagram of E.coli TOP10-pDawn(cI-LVA)-RBS070-LKD-T1.

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] [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).