Difference between revisions of "Part:BBa K3020000:Design"

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The recA promoter does not have a strict sequence range on the NCBI website. Therefore, when using the PCR to clone the target fragment, we designed the primer and completely cloned the recA repair protein base ATG upstream of 390 bp.
 
The recA promoter does not have a strict sequence range on the NCBI website. Therefore, when using the PCR to clone the target fragment, we designed the primer and completely cloned the recA repair protein base ATG upstream of 390 bp.
 +
 +
The cloned positive plasmid was sent to the company for sequencing, and the sequencing primers were VF2 and VR. The sequencing results are shown in the figure. It can be seen that the plasmid sequence is consistent with the theoretical recA+eGFP sequence, indicating that the plasmid was successfully constructed.
 +
[[File:BIT201905-pre-cexu.png|frame|center|'''Sequencing map''']]
  
 
===Source===
 
===Source===
  
  
−
  The recA promoter fragment was cloned from the genome of E.coli MG1655 strain as a template, and ligated upstream of the reporter gene by enzyme cleavage and other molecular cloning methods, and the target fragment was cloned into the standard vector pSB1A2.
+
The recA promoter fragment was cloned from the genome of E.coli MG1655 strain as a template, and ligated upstream of the reporter gene by enzyme cleavage and other molecular cloning methods, and the target fragment was cloned into the standard vector pSB1A2.
  
−
  The eGFP gene sequence used in this experiment was obtained from NCBI, sequence ID: 20473140. Due to the preference of codons, the level of foreign gene expression was affected, and the gene sequence was given to Jin Weizhi (Su State) Bio Co., Ltd. performs codon optimization for E. coli hosts.
+
The eGFP gene sequence used in this experiment was obtained from NCBI, sequence ID: 20473140. Due to the preference of codons, the level of foreign gene expression was affected, and the gene sequence was given to Jin Weizhi (Su State) Bio Co., Ltd. performs codon optimization for E. coli hosts.
  
 
===References===
 
===References===

Latest revision as of 07:26, 10 September 2019


Green fluorescent protein reporter gene under oxidative damage-sensitive initiation


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 812
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The recA promoter does not have a strict sequence range on the NCBI website. Therefore, when using the PCR to clone the target fragment, we designed the primer and completely cloned the recA repair protein base ATG upstream of 390 bp.

The cloned positive plasmid was sent to the company for sequencing, and the sequencing primers were VF2 and VR. The sequencing results are shown in the figure. It can be seen that the plasmid sequence is consistent with the theoretical recA+eGFP sequence, indicating that the plasmid was successfully constructed.

Sequencing map

Source

The recA promoter fragment was cloned from the genome of E.coli MG1655 strain as a template, and ligated upstream of the reporter gene by enzyme cleavage and other molecular cloning methods, and the target fragment was cloned into the standard vector pSB1A2.

The eGFP gene sequence used in this experiment was obtained from NCBI, sequence ID: 20473140. Due to the preference of codons, the level of foreign gene expression was affected, and the gene sequence was given to Jin Weizhi (Su State) Bio Co., Ltd. performs codon optimization for E. coli hosts.

References