Difference between revisions of "Part:BBa K3645011"
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===Characterization from iGEM21-NNU-China===
 
===Characterization from iGEM21-NNU-China===
  
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;MazF is toxin protein in MazF-MazE, toxin-antitoxin module. It was first registered in 2013 and used as an mRNA endonuclease. It kills bacteria without cracking them, which will directly influence the value of OD<sub>600</sub>.  
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Cytosine base editors (CBEs) enable targeted C•G-to-T•A conversions in genomic DNA, consisting of dSpCas9, CDA, and UGI [1]. It was first registered in 2020. In order to test the editing efficiency of this composite part, we construct the dual plasmid system based on the BBa_K3645011. We selected the cadA, maeA, and maeB genes as the testing sites, and the related pTarget plasmids were constructed. Results showed that the (<partinfo>BBa_K3645011</partinfo>) can successfully work in the BL21 (DE3), and the editing efficiency of single gene editing, double genes editing and triple genes editing can reach 85%, 56% and 25%, respectively (Fig. 1). These results provide references for future iGEM teams to choose gene-editing tools in E.coli.
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;In order to quantify the toxicity of mazF, colony forming unit (CFU) cell viability assays were used to measure functionality of the circuit(1).
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Because direct expression of MazF will kill bacteria, we construct the circuit "P<sub>Bad/araC</sub>-RBS-MazF-terminator-pSB1C3"(<partinfo>BBa_K3332083</partinfo>) and "P<sub>Bad/araC</sub>-RBS-EYFP-terminator-pSB1C3"(<partinfo>BBa_K3332082</partinfo>) in ''E.coli'' BL21(DE3) to characterize its function.  
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:'''Fig.1''' genetic circuit of P<sub>Bad/araC</sub>-RBS-MazF-terminator.
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:'''Fig.1''' The gene editing efficiency of the part of dCas9-CDA-UGI.
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;When 0.2% arabinose was added, the experimental group showed a sharp decrease on the CFU, which indicates the protein encoded by MazF is fatal to ''E. coli''. It further demonstrated that the MazF encoded by J23100-RBS (<partinfo>BBa_K880005</partinfo>) was able to work as a toxin.
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        <img src="https://2020.igem.org/wiki/images/c/c7/T--XMU-China--XMU-China_2020_BM-CFU.png" width="45%" style="float:center">
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:'''Fig.2''' The results of CFU.The round dot indicates the non induction group, and the square dot indicates the induction group.
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Revision as of 13:31, 17 October 2021


Target-AID (CBE)

Contains the full CDS of Target-AID, whose Cas9 part was replace with our lab's dCas9.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1099
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 4775
    Illegal BamHI site found at 3378
    Illegal XhoI site found at 4384
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Contribution From XMU-China 2020

Group: iGEM Team NNU-China 2021

Author: Yan Xu

Summary: Testing its gene editing efficiency in BL21 (DE3)

Characterization from iGEM21-NNU-China

        Cytosine base editors (CBEs) enable targeted C•G-to-T•A conversions in genomic DNA, consisting of dSpCas9, CDA, and UGI [1]. It was first registered in 2020. In order to test the editing efficiency of this composite part, we construct the dual plasmid system based on the BBa_K3645011. We selected the cadA, maeA, and maeB genes as the testing sites, and the related pTarget plasmids were constructed. Results showed that the (BBa_K3645011) can successfully work in the BL21 (DE3), and the editing efficiency of single gene editing, double genes editing and triple genes editing can reach 85%, 56% and 25%, respectively (Fig. 1). These results provide references for future iGEM teams to choose gene-editing tools in E.coli.

Fig.1 The gene editing efficiency of the part of dCas9-CDA-UGI.