Difference between revisions of "Part:BBa K5246044"

Revision as of 06:55, 29 September 2024

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

Introduction

Usage and Biology

This is a part of the complete holdfast polymerization and export apparatus BBa_K5246046 used in Vilnius-Lithuania iGEM 2024 project "Synhesion" https://2024.igem.wiki/vilnius-lithuania/. This part can also be used separately for polysaccharide export, but this feature needs more characterization.

Sequence and Features

Functional Parameters

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.

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.

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.

References