Part:BBa_K2585000:Design

Eukaryotic aspartyl protease family protein

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 25
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

To construct our part(BBa_K2585000), we did following work.

First, we did codon optimization for our target gene pcs1 from Arabidopsis thaliana and we named it copcs1. However, there are three illegal enzyme sites in the optimization sequence, as is shown in the Fig.1 below. So then, we exchange the site sequence accoding to the degeneracy of codons. The changes are shown in the Fig.2 below. Fig.1 Three illegal enzyme sites in copcs1. Fig.2 Illegal enzyme sites-free in new sequence.

To accomplish the goal, we design the primers in the chart. And we conducted the experiments as below.

First, we used the primers(F-xbaⅠ-pcs1 & R-speⅠ-pcs1) below to copy copcs1 gene by PCR as well as to add XbaⅠand SpeⅠenzyme sites respectively on both ends. Next, again through PCR with primers(F-xbaⅠ-pcs1 & R-suffix), so as to add pstⅠenzyme sites outside the speⅠ’s. And then, gaining the target fragment after purification.

Then using XbaⅠand PstⅠenzyme to digest both the copcs1 PCR-purified products and pSB1C3 plasmid backbone. After enzyme digestion, they were purified respectively. Finally, after fragments ligation, transformation, colony PCR, sequencing, bacteria culture and plasmid extraction, our part(BBa_K2585000) was successfully constructed.

Source

NCBI Gene ID: 831845