Difference between revisions of "Part:BBa K3875027"

 
 
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===Usage and Biology===
 
===Usage and Biology===
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<strong><h2>1.Design</strong></h2><br>
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We designed the part(BBa_K3875027) to characterize <i>mazE</i> and <i>mazF</i>.
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We performed experiment to work our final genetic circuits and characterized <i>mazE</i> and <i>mazF</i>.<br>
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This experiment consists of the four parts below:<br>
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1.Obtain the mazEF gene and the vgb gene<br>
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2.Build a gene expression vector<br>
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3.Import the gene expression vector to detect the properties of engineered bacteria<br>
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<img src="https://static.igem.org/mediawiki/parts/b/bb/T-buct-vgb-4655.png"width="640px";height="30px"/><br>
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<strong><h2>2.Methods</strong></h2>
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1.Design primers with a homologous arm, using pcr to obtain the target genes <i>mazEF</i> and vgb from <i>E. coli BW25113</i><br>
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2.The expression vector pZE is constructed by the method of enzymatic enzyme connection<br>
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3.Validate build results and sequencing<br>
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4.Import the constructed expression vector into <i>nissle 1917</i> observation<br>
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<strong><h2>3.Results<br></strong></h2>
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After the culture and sequencing verification of pZE-pvgb, the constructed plasmids will be imported into <i>nissle 1917</i>, tighten the lid to ensure low oxygen status, 37 degrees Celsius shaker culture 12-16h, found that the colonies grow and the size is obvious, after the extraction of plasmids sent sequencing found lac promoter loss, indicating that the construction of suicide system is not successful<br>
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<img src="https://static.igem.org/mediawiki/parts/9/9d/T—BUCT—pae-maze.jpg"width="640px";height="30px"/><br>
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Figure. The growth of bacteria imported into the suicide system<br>
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Latest revision as of 16:38, 20 October 2021


Usage and Biology

1.Design


We designed the part(BBa_K3875027) to characterize mazE and mazF. We performed experiment to work our final genetic circuits and characterized mazE and mazF.
This experiment consists of the four parts below:
1.Obtain the mazEF gene and the vgb gene
2.Build a gene expression vector
3.Import the gene expression vector to detect the properties of engineered bacteria

2.Methods

1.Design primers with a homologous arm, using pcr to obtain the target genes mazEF and vgb from E. coli BW25113
2.The expression vector pZE is constructed by the method of enzymatic enzyme connection
3.Validate build results and sequencing
4.Import the constructed expression vector into nissle 1917 observation

3.Results

After the culture and sequencing verification of pZE-pvgb, the constructed plasmids will be imported into nissle 1917, tighten the lid to ensure low oxygen status, 37 degrees Celsius shaker culture 12-16h, found that the colonies grow and the size is obvious, after the extraction of plasmids sent sequencing found lac promoter loss, indicating that the construction of suicide system is not successful

Figure. The growth of bacteria imported into the suicide system

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 450
    Illegal EcoRI site found at 665
    Illegal XbaI site found at 87
    Illegal XbaI site found at 941
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 450
    Illegal EcoRI site found at 665
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 450
    Illegal EcoRI site found at 665
    Illegal BamHI site found at 738
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 450
    Illegal EcoRI site found at 665
    Illegal XbaI site found at 87
    Illegal XbaI site found at 941
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 450
    Illegal EcoRI site found at 665
    Illegal XbaI site found at 87
    Illegal XbaI site found at 941
  • 1000
    COMPATIBLE WITH RFC[1000]