Difference between revisions of "Part:BBa K3409011"

 
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Contains the elements necessary to express the fourth gene from the Microcin PDI gene cluster (mcpD: a homolog of hlyD, coding for part of the secretion system). The sequence is organized as follows: a promoter (BBa_J23118), an RBS (BBa_J61118), the CDS for McpD (BBa_K3409006). Will constitutively generate McpD. For appropriate secretion, it must be followed by BBa_K3409012, which contains the CDS for McpB.  
 
Contains the elements necessary to express the fourth gene from the Microcin PDI gene cluster (mcpD: a homolog of hlyD, coding for part of the secretion system). The sequence is organized as follows: a promoter (BBa_J23118), an RBS (BBa_J61118), the CDS for McpD (BBa_K3409006). Will constitutively generate McpD. For appropriate secretion, it must be followed by BBa_K3409012, which contains the CDS for McpB.  
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===Characterization===
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NOTE: In this section: BB1 = part BBa_K3409010  -  BB2 = part BBa_K3409011 – BB3 = part BBa_K3409012
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PCR AMPLIFICATION OF PARTS
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[[File: PCR_BB1_BB2_BB3.png|border|700px]]
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Once received, the synthetized fragments are resuspended and amplified via polymerase chain reaction (PCR) to obtain a higher quantity of DNA. Every PCR sample is verified by gel electrophoresis to analyze the PCR results. First, the appropriate bands for BioBrick 1 (1.5 kb) , BioBrick 2 (1.4 kb) and BioBrick 3 (2.4 kb) amplified with the primers for Hifi Assembly method, are present at their expected size (Figure 1A).
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Then, the appropriate bands for BioBrick 1 (1.5 kb), BioBrick 2 (1.4 kb) and BioBrick 3 (2.4 kb) amplified with the primers for BioBrick Assembly method, are present at their expected size on Figure 1B.
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[[File: Bb1bb2.png|border|700px]]
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A proportional amount of white colonies compared to different ligation ratios can be observed on the plates (Figure 2A) and no colonies are counted on the control plate. Thus, the transformation seems to have worked. Regarding the verification of the ligation, colonies needed to be tested after having completed a miniprep procedure to obtain the plasmid. The extracted plasmids of the colonies have been cut with restriction enzymes (RE) BgII and NdeI. The expected bands are depicted on Figure 2C. Sample of lane 1, 3 and 6 , corresponding to colony N°1, colony N°3 and colony N°6, seem to have the 2 corresponding band size.
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This workflow was stopped at this step of proceeding for a matter of time. However, the next step would have been the assembly with the obtained plasmid of colonies 1, 3 and 6, with BioBrick and after verification, continue with the verification procedures (Agar diffusion assay and more).
  
 
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Latest revision as of 21:08, 25 October 2020


Microcin PDI cluster gene for McpD

Contains the elements necessary to express the fourth gene from the Microcin PDI gene cluster (mcpD: a homolog of hlyD, coding for part of the secretion system). The sequence is organized as follows: a promoter (BBa_J23118), an RBS (BBa_J61118), the CDS for McpD (BBa_K3409006). Will constitutively generate McpD. For appropriate secretion, it must be followed by BBa_K3409012, which contains the CDS for McpB.

Characterization

NOTE: In this section: BB1 = part BBa_K3409010 - BB2 = part BBa_K3409011 – BB3 = part BBa_K3409012

PCR AMPLIFICATION OF PARTS

PCR BB1 BB2 BB3.png

Once received, the synthetized fragments are resuspended and amplified via polymerase chain reaction (PCR) to obtain a higher quantity of DNA. Every PCR sample is verified by gel electrophoresis to analyze the PCR results. First, the appropriate bands for BioBrick 1 (1.5 kb) , BioBrick 2 (1.4 kb) and BioBrick 3 (2.4 kb) amplified with the primers for Hifi Assembly method, are present at their expected size (Figure 1A). Then, the appropriate bands for BioBrick 1 (1.5 kb), BioBrick 2 (1.4 kb) and BioBrick 3 (2.4 kb) amplified with the primers for BioBrick Assembly method, are present at their expected size on Figure 1B.

Bb1bb2.png

A proportional amount of white colonies compared to different ligation ratios can be observed on the plates (Figure 2A) and no colonies are counted on the control plate. Thus, the transformation seems to have worked. Regarding the verification of the ligation, colonies needed to be tested after having completed a miniprep procedure to obtain the plasmid. The extracted plasmids of the colonies have been cut with restriction enzymes (RE) BgII and NdeI. The expected bands are depicted on Figure 2C. Sample of lane 1, 3 and 6 , corresponding to colony N°1, colony N°3 and colony N°6, seem to have the 2 corresponding band size.

This workflow was stopped at this step of proceeding for a matter of time. However, the next step would have been the assembly with the obtained plasmid of colonies 1, 3 and 6, with BioBrick and after verification, continue with the verification procedures (Agar diffusion assay and more).

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]