Part:BBa_K5246044

C.Crescentus CB2/CB2A hfsA-hfsB-hfsD Part of polysaccharide export apparatus

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1740
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1740
  Illegal NotI site found at 475
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1740
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1740
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1740
  Illegal NgoMIV site found at 151
  Illegal NgoMIV site found at 343
  Illegal NgoMIV site found at 352
  Illegal NgoMIV site found at 924
  Illegal NgoMIV site found at 1087
  Illegal NgoMIV site found at 2026
  Illegal NgoMIV site found at 2672
  
• 1000
  COMPATIBLE WITH RFC[1000]

Experimental characterization

Part cloning

All of the proteins composing this system are responsible for polysaccharide polymerization and export. Since the system's proteins are found in the membrane, we concluded that using a low-copy plasmid would decrease the probability of inclusion body formation. Their formation would diminish the functionality of our system, as the proteins would not allow the polysaccharide to be exported outside the bacteria.

To assemble specifically this part into BBa_K5246044 to then further assemble the holdfast synthesis pathway in E. coli , we had to assemble this part first into a backbone of pACYC-Duet-1 with other BBa_K5246044 genes. We designed a strategy to maximize the success of plasmid assembly by first assembling plasmids with 3 genes and, after verifying the sequences, integrating 3 left genes into that backbone (Fig. 2). In this way, we prevented Golden Gate assembly errors by trying to construct plasmids from 8 or more fragments.

Fig. 2. Plasmid construction strategy. Plasmids are constructed in two rounds, cloning 3 genes at a time. Verified by colony PCR, restriction digestion analysis, and Nanopore sequencing

The assembly was done using Golden Gate assembly with IIS AarI restriction enzyme sites introduced during PCR amplification. The backbone of pACYC-Duet-1 (Novagen) and fragments were amplified using Phusion Plus DNA polymerase, as the genome of C. crescentus has a high GC% content making the appearance of non-specific products during PCR amplification more common and primer design more challenging (Fig. 3). Since, hfsA gene had an AarI RE site directly in the gene, this site was domesticated during side directed mutagenesis.

Fig. 3 Plasmid construction strategy. Plasmids are constructed in two rounds, cloning 3 genes at a time. Verified by colony PCR, restriction digestion analysis, and Nanopore sequencing

Due to the high amount of non-specific products, the fragments were gel-purified. Vectors and fragments composing this operon, were mixed in equimolar amounts with GG reaction components and incubated as described in protocol. The reaction was later transformed into E. coli Mach1 (Thermo Scientific) competent cells. The assembly was then confirmed with restriction digest analysis (Fig. 4) and positive colonies were sequenced.

Fig. 4 Restriction digest analysis of C. crescentus CB2 pACYC-hfsA-hfsB-hfsD. On the left - expected in silico profile of restriction digest with EcoRI and ScaI, on the right - digested plasmids - 1-6 colonies, M - molecular weight ladder, GeneRuler DNA Ladder Mix (Thermo Scientific)