Part:BBa_K4907040

mn-SOD-ccdA-sgRNA sequence

Biology

ccdA is the gene found within the ccd operon, encoding the antidote protein (CcdA) that protects cells from the toxic effects of CcdB. Mn-SOD, a subtype of SOD, utilizes manganese (Mn) as a cofactor, existing in the form of a homotetramer. The expression of Mn-SOD gene in several insect species increased their survival at low temperatures (1). The gRNA is a short synthetic RNA composed of a scaffold sequence necessary for Cas-binding and a user-defined 20 nucleotide spacer (called N20 sequence) that decides the target genes to be modified. By changing the N20 sequence, one can change the gene target of the Cas protein.

Usage and design

For the biosafety and stress resistance of our project, we need to transfer the ccdA gene and the Mn-SOD gene into the DH10β genome, where the rne is located. We get our donor fragment by PCR.

Characterization

Agarose gel electrophoresis (AGE)

We linked the rne fragment of DH10β to the ccdA gene and the Mn-SOD gene through multiple rounds of PCR. At a position around 2098bp, a target band of approximately 3000bp was observed (Fig. 1).

Fig. 1 DNA gel electrophoresis of the PCR products of BBa_K4907040

Reference

1. Y. I. Kim et al. Modulation of MnSOD protein in response to different experimental stimulation in Hyphantria cunea. Comp Biochem Physiol B Biochem Mol Biol. 157, 343–350 (2010).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]