Part:BBa_K5226077

gRNA-△cysNC-key

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1852
  Illegal XbaI site found at 98
  Illegal PstI site found at 1474
  Illegal PstI site found at 2047
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1852
  Illegal NheI site found at 765
  Illegal PstI site found at 1474
  Illegal PstI site found at 2047
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1852
  Illegal BamHI site found at 92
  Illegal BamHI site found at 1002
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1852
  Illegal XbaI site found at 98
  Illegal PstI site found at 1474
  Illegal PstI site found at 2047
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1852
  Illegal XbaI site found at 98
  Illegal PstI site found at 1474
  Illegal PstI site found at 2047
  Illegal NgoMIV site found at 364
  
• 1000
  COMPATIBLE WITH RFC[1000]

Introduction

The existing methods for large-scale production of P34HB primarily rely on microbial fermentation. A key limiting factor in this process is the molar ratio of 4HB. Increasing the 4HB molar ratio can lead to a decrease in the melting temperature and apparent fusion heat of the copolymer, as well as an improvement in the polymer's deformation resistance. Therefore, enhancing the molar ratio of 4HB is crucial for the modification of P34HB.

Usage and Biology

To stabilize cell dry weight while increasing the 4HB molar ratio, we investigated the effect of the pSEVA321 on cell dry weight. We hypothesized that the addition of chloramphenicol during the fermentation process impacted cell growth. Consequently, we decided to adjust the screening pressure and knock out cysNC, a gene encoding a key enzyme in the sulfate assimilation pathway, in Halomonas TD to block the supply of sulfur sources for methionine synthesis. Simultaneously, we incorporated the cysNC gene into the pSEVA321 backbone to screen for strains that had been successfully transformed with the 321-porin194-4hbd-sucD-ogdA-porin194-orfZ plasmid.
We used CRISPR/Cas9 for gene knockout. Due to the challenges of gene editing in Halomonas TD, we employed two methods to block the expression of cysNC. The first method involved knocking out all codons from the start codon (ATG) to the stop codon. The second approach entailed knocking out the core region from its full-length sequence. We designed two sets of gRNAs targeting the specific regions for each knockout method. Both methods were performed simultaneously, and successful knockouts were selected for the subsequent fermentation step.

Experimental characterisation

Conjugation

Plasmids were transferred from E. coli S17-1 to H. bluephagenesis TD80 using an optimized conjugation transformation protocol as follows: E. coli S17-1 as the donor cell which harbors the plasmid was cultured in LB medium in the presence of appropriate antibiotic(s). H. bluephagenesis TD80 or recombinant H. bluephagenesis TD80 were the recipient cells, and were cultured in 60LB. Select monoclonal antibodies and culture them overnight in 5 mL of the appropriate medium. Next, take 20 µL of both the recipient bacterial solution and the donor bacterial solution, and mix them on a 20 LB plate. Incubate the mixture overnight at 37 °C. Finally, resulting bacterial lawn was re-suspended in 60LB medium and spread on a 60LB agar plate with appropriate antibiotics, followed by incubation at 37 °C for 24–48 h to select for transconjugants.

Initially, S17-1 (pSEVA321-Cas9) was conjugated with TD80 and subsequently cultured on a 60AC plate containing ampicillin and chloramphenicol. After achieving monoclonal growth, we obtained TD80 (Cas9) by streaking on the 60AC plate. Following the acquisition of TD80 (Cas9), it was conjugated with S17-1 (pSEVA341 gRNA) and cultured on a 60CSM plate containing chloramphenicol, spectinomycin, and methionine. Once monoclonal antibodies grew, we can verify the successful knockout of cysNC or its core region

PCR validation

After growing monoclonal colonies on a 60CSM plate containing chloramphenicol, spectinomycin, and methionine, we designed a pair of universal primers at 50-100bp around the homologous arm of the genome, along with a specific primer located in the middle of the target knockout gene. First, we performed colony PCR using the universal primers and sampled the bacteria on a 60LB plate. If the knockout was successful, we expected to see a band around 4500 bp; if it failed, the band would be around 2200 bp. On the following day, we conducted a secondary validation on the successfully knocked-out strains using the specific primers. If the knockout was successful, no bands would be present; if it failed, a band approximately 2000 bp in length would appear.

Eliminate resistance

After successful double validation, in order to facilitate subsequent gene editing and plasmid transfer of the strain, we used a deep well plate to eliminate resistance in the strain at a temperature of 42 °C. The deep well plate consisted of 1 ml of 60LB and 0.2 g/L methionine, and the cultures were incubated for 24 hours. Starting from the third generation, we verified whether resistance had been removed. Since pSEVA321-Cas9 is easily eliminated, our primary focus was to verify whether pSEVA341-gRNA-cysNC had been removed. We took 5 μL of the bacterial solution and transferred it to a 60ASM plate containing spectinomycin and methionine, followed by incubation at 37 °C for 12 hours. If no colonies grew, this indicated that resistance had been successfully removed.
The next generation was then streaked to select monoclonal colonies from the 60M plate containing methionine for double validation. If successful, the bacteria could be preserved; if not, it would indicate that the bacteria on the sample plate had reverted to the wild type. In such a case, we needed to re-verify whether the bacteria on the sample plate had returned to wild type. If they had, the coated plate should be reconnected; if there was no response, we would begin the de-antibody passage and repeat the above steps until success was achieved.

Sequencing validation

The sequencing outcomes revealed that the core region of the cysNC had been successfully knocked out.

We can now proceed to the next step of the experiment, which involves constructing cysNC on pSEVA321 as a new selection pressure. For further experiments, please refer to <a href="https://parts.igem.org/Part:BBa_K5226078">BBa_K5226078</a>.