Difference between revisions of "Part:BBa K5133000"

 
 
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<partinfo>BBa_K5133000 short</partinfo>
 
<partinfo>BBa_K5133000 short</partinfo>
  
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TBD
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Group: <b>GEC-China (iGEM 2024, team number: #5133)</b>
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==<b>Brief introduction</b>==
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This basic part is derived from plasmid pJL1 (Addgene: #69496)<sup>[1]</sup>, including a conserved DNA sequence of T7 promoter as <b>5'-taatacgactcactatagggaga-3'</b><sup>[2]</sup>. The plasmid pJL1 is commonly used for the <i>in vitro</i> sfGFP expression of cell-free protein synthesis (CFPS)<sup>[3]</sup>. Hence, this part is used for the construction of three composite parts: <bbpart>BBa_K5133004</bbpart> (sfGFP generator), <bbpart>BBa_K5133006</bbpart> (Microcin H47 generator), and <bbpart>BBa_K5133008</bbpart> (Microcin M generator), for CFPS in our project.
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==<b>Design and characterization</b>==
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The plasmid design of this biological part is shown as <b>Figure 1</b>, assembled with iGEM standard backbone <bbpart>pSB1C3</bbpart>. Result of Sanger sequencing shows the correct construction of this part (<b>Figure 2</b>).
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<center>
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<html lang="en">
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<head>
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    <meta charset="UTF-8">
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    <meta name="viewport" content="width=device-width, initial-scale=1.0">
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    <title>Resizable Image</title>
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    <style>
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        .resizable-img1 {
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            max-width: 60%;
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            height: auto;
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        }
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    </style>
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</head>
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<body>
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    <img src="https://static.igem.wiki/teams/5133/bba-k5133000-1.jpg"  class="resizable-img1">
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</body>
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</html>
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</center>
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<center><b>Figure 1. Schematic design of this part, generated by SnapGene.</b></center>
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<center>
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<html lang="en">
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<head>
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    <meta charset="UTF-8">
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    <meta name="viewport" content="width=device-width, initial-scale=1.0">
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    <title>Resizable Image</title>
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    <style>
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        .resizable-img2 {
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            max-width: 90%;
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            height: auto;
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        }
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    </style>
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</head>
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<body>
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    <img src="https://static.igem.wiki/teams/5133/bba-k5133000-2.jpg"  class="resizable-img2">
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</body>
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</html>
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</center>
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<center><b>Figure 2. Validation of DNA sequence by Sanger sequencing, generated by SnapGene.</b></center>
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==<b>Usages</b>==
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This part is used for the construction of three composite parts: <bbpart>BBa_K5133004</bbpart> (sfGFP generator), <bbpart>BBa_K5133006</bbpart> (Microcin H47 generator), and <bbpart>BBa_K5133008</bbpart> (Microcin M generator), for CFPS in our project.
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==<b>DNA sequence (from 5' to 3')</b>==
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atcccgcgaaat<font color="red">taatacgactcactatagggaga</font>ccacaacggtttccctctagaaataattttgtttaacttt
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<font color="red">Red font: conserved T7 promoter</font>
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==<b>References</b>==
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[1] https://www.addgene.org/69496/
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[2] Conrad, T. et al. Maximizing transcription of nucleic acids with efficient T7 promoters. <b>Communications Biology</b> 3, 439 (2020). doi: 10.1038/s42003-020-01167-x
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[3] Ba, F. et al. Expanding the toolbox of probiotic <i>Escherichia coli</i> Nissle 1917 for synthetic biology. <b>Biotechnology Journal</b> 19, 2300327 (2024). doi: 10.1002/biot.202300327
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 06:22, 27 July 2024


T7 promoter (from plasmid pJL1)

Group: GEC-China (iGEM 2024, team number: #5133)


Brief introduction

This basic part is derived from plasmid pJL1 (Addgene: #69496)[1], including a conserved DNA sequence of T7 promoter as 5'-taatacgactcactatagggaga-3'[2]. The plasmid pJL1 is commonly used for the in vitro sfGFP expression of cell-free protein synthesis (CFPS)[3]. Hence, this part is used for the construction of three composite parts: BBa_K5133004 (sfGFP generator), BBa_K5133006 (Microcin H47 generator), and BBa_K5133008 (Microcin M generator), for CFPS in our project.


Design and characterization

The plasmid design of this biological part is shown as Figure 1, assembled with iGEM standard backbone pSB1C3. Result of Sanger sequencing shows the correct construction of this part (Figure 2).


Resizable Image


Figure 1. Schematic design of this part, generated by SnapGene.



Resizable Image


Figure 2. Validation of DNA sequence by Sanger sequencing, generated by SnapGene.




Usages

This part is used for the construction of three composite parts: BBa_K5133004 (sfGFP generator), BBa_K5133006 (Microcin H47 generator), and BBa_K5133008 (Microcin M generator), for CFPS in our project.


DNA sequence (from 5' to 3')

atcccgcgaaattaatacgactcactatagggagaccacaacggtttccctctagaaataattttgtttaacttt

Red font: conserved T7 promoter


References

[1] https://www.addgene.org/69496/

[2] Conrad, T. et al. Maximizing transcription of nucleic acids with efficient T7 promoters. Communications Biology 3, 439 (2020). doi: 10.1038/s42003-020-01167-x

[3] Ba, F. et al. Expanding the toolbox of probiotic Escherichia coli Nissle 1917 for synthetic biology. Biotechnology Journal 19, 2300327 (2024). doi: 10.1002/biot.202300327


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 51
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 51
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 51
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 32