Difference between revisions of "Part:BBa K4325021"
 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K4325021 short</partinfo>
 
<partinfo>BBa_K4325021 short</partinfo>
 
===Description===
 
===Description===
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This composite part is a generator consisting of pDawn(cI-LVA)(<partinfo>BBa_K1075044</partinfo>) and X174E(<partinfo>BBa_K1835500</partinfo>).
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This composite part is a generator consisting of pDawn(cI-LVA) (<partinfo>BBa_K1075044</partinfo>) ,X174E (<partinfo>BBa_K1835500</partinfo>) and T1 terminator (<partinfo>BBa_K3033016</partinfo>).
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[[File:pDawn.png|600px|thumb|center|Figure 1: Gene circuit of pDawn(CI-LVA)-X174E-T1.]] <p></p>
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[[File:pDawn.png|600px|thumb|center|Figure 1: Genetic circuit of pDawn(cI-LVA)-X174E-T1.]] <p></p>
 
===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 X174E (<partinfo>BBa_K1835500</partinfo>) into the pSEVA331 expression vector, which was inserted into <i>E. coli</i> TOP10 and screened for colonies that grew in the dark but did not grow under blue light. Finally, the pSEVA331-pDawn (cI-LVA) -X174E-T1 plasmids were selected and further inserted into <i>G. hansenii</i>  ATCC53582 to verify the responsiveness of pDawn (CI-LVA) to blue light.
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<p>We inserted the blue light responsive system pDawn(cI-LVA) (<partinfo>BBa_K1075044</partinfo>) and the lysis gene X174E (<partinfo>BBa_K1835500</partinfo>) into the pSEVA331 expression vector, which was introduced into <i>E. coli</i> TOP10 and screened for colonies that grew in the dark but did not grow under blue light.</p>
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</p>
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===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 pSEVA331-pDawn(cI-LVA)-X174E-T1 in response to blue light lysis in <i>E. coli</i>.</h4>
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<h4>1.Batch screening of pDawn-X174E-T1 by evaluating the response to blue light. </h4>
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[[File:PDawn1.png|600px|thumb|center|Figure 2: Growth condition of pSEVA331-pDawn (cI-LVA) -LKD-T1-TOP10 in the dark and under light.]]<p></p>
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[[File:K21 2.png|600px|thumb|center|Figure 2: Growth condition of <i>E. coli</i> TOP10-pDawn(cI-LVA)-X174E-T1-TOP10 in the dark and under light.]]<p></p>
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[[File:PDawn2.png|600px|thumb|center|Figure 3: Growth curve diagram  of pSEVA331-pDawn (cI-LVA) -X174E-T1-pSEVA331-TOP10.]]<p></p>
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[[File:K21 3.png|600px|thumb|center|Figure 3: Growth curve diagram  of <i> E. coli</i> TOP10-pDawn(cI-LVA)-X174E-T1-TOP10.]]<p></p>
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<p>As shown in Figure 2, the colony numbers 1st, 4<sub>th</sub> , 5<sub>th</sub>, 6<sub>th</sub>, 7<sub>th</sub>, 8<sub>th</sub>, 10<sub>th</sub>, 14<sub>th</sub>, and 16<sub>th</sub> met our expectation. To further searching the lysis effect of pDawn (cI-LVA) -X174E-T1-pSEVA331-TOP10, we measured the OD600 value of pSEVA331-pDawn (cI-LVA) -X174E-T1-TOP10 periodically and ploting the growth curve diagram. As shown in Figure 3, the lysis effect of pSEVA331-pDawn (cI-LVA) -X174E-T1-TOP10 is mediocre.</p>
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<p>As shown in Figure 2, the 1<sup>th</sup> , 4<sup>th</sup> , 5<sup>th</sup>, 6<sup>th</sup>, 7<sup>th</sup>, 8<sup>th</sup>, 10<sup>th</sup>, 14<sup>th</sup>, and 16<sup>th</sup> colonies met our expectations. To further search the lysis effect of pDawn(cI-LVA) -X174E-T1, we tracked the OD<sub>600</sub> value of <i>E. coli</i>  TOP10-pSEVA311-pDawn(cI-LVA)-X174E-T1 and plotted the growth curves diagram as shown in Figure 3. It shows that the lysis effect of pDawn(cI-LVA)-X174E-T1 is mediocre.</p>
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<h4>2. pDawn(cI-LVA)-X174E-T1-pSEVA331 in response to blue photolysis in <i> G. hansenii</i> ATCC53582.</h4>
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[[File:K27 5.png|600px|thumb|center|Figure3: Successfully identified by agarose 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>(4)pSEVA331-pDawn(cI-LVA)-X174E-T1- <i>G. hansenii</i>  ATCC53582 and all of them were constructed successfully.]]
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<p>We transformed the pDawn (cI-LVA) -X174E-T1 plasmid into G. hansenii ATCC53582 and identified pDawn (cI-LVA) -X174E-T1 by using agarose gel electrophoresis. As shown in Figure 3, we successfully constructed the pDawn (cI-LVA) -X174E-T1.</p>
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<h3>References</h3>
 
<h3>References</h3>
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<p>Robert Ohlendorf 1 , Roee R. Vidavski 2 , Avigdor Eldar 2, Keith Moffat 3, 4 and Andreas Möglich 1, 3.From Dusk till Dawn: One-Plasmid Systems for Light-Regulated Gene Expression.</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. 416, 534-542 (2012).</p>
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<p>[2] Witte, A., Wanner, G., Sulzner, M. & Lubitz, W. Dynamics of PhiX174 protein E-mediated lysis of <i>Escherichia coli</i>. Arch. Microbiol. 157, 381-8 (1992).</p>

Latest revision as of 14:34, 12 October 2022

pDawn-X174E-T1

Description

This composite part is a generator consisting of pDawn(cI-LVA) (BBa_K1075044) ,X174E (BBa_K1835500) and T1 terminator (BBa_K3033016).

Figure 1: Genetic circuit of pDawn(cI-LVA)-X174E-T1.

Usage

We inserted the blue light responsive system pDawn(cI-LVA) (BBa_K1075044) and the lysis gene X174E (BBa_K1835500) into the pSEVA331 expression vector, which was introduced into E. coli TOP10 and screened for colonies that grew in the dark but did not grow under 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-X174E-T1 by evaluating the response to blue light.

Figure 2: Growth condition of E. coli TOP10-pDawn(cI-LVA)-X174E-T1-TOP10 in the dark and under light.

Figure 3: Growth curve diagram of E. coli TOP10-pDawn(cI-LVA)-X174E-T1-TOP10.

As shown in Figure 2, the 1th , 4th , 5th, 6th, 7th, 8th, 10th, 14th, and 16th colonies met our expectations. To further search the lysis effect of pDawn(cI-LVA) -X174E-T1, we tracked the OD600 value of E. coli TOP10-pSEVA311-pDawn(cI-LVA)-X174E-T1 and plotted the growth curves diagram as shown in Figure 3. It shows that the lysis effect of pDawn(cI-LVA)-X174E-T1 is mediocre.

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] Witte, A., Wanner, G., Sulzner, M. & Lubitz, W. Dynamics of PhiX174 protein E-mediated lysis of Escherichia coli. Arch. Microbiol. 157, 381-8 (1992).