Part:BBa_K5226074

B0064-porin281-Vib-ftl-porin194-Vib-folD

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1189
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1189
  Illegal NheI site found at 2375
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1189
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1189
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1189
  Illegal NgoMIV site found at 455
  
• 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.

Prior to embarking on this project, our laboratory had already conducted research on the production of P34HB. It was found that the expression of the 4hbd-sucD-ogdA-orfZ gene cluster could increase the molar ratio of 4HB. Following fermentation using Mmp1 inducible promoter, the porin194 constitutive promoter was considered more suitable based on the concentration gradient induction trend observed with IPTG.

Since two plasmids, pSEVA321 and pSEVA341, are commonly used in the laboratory, the gene cluster has only been previously expressed through the pSEVA321 plasmid. Our intention is to introduce the porin194-4hbd-sucD-ogdA-porin194-orfZ gene cluster into TD80 to synthesize P34HB, utilizing both the pSEVA341 and pSEVA321 plasmids, which allow us to evaluate which plasmid yields better results.

Usage and Biology

Although TD80 possesses its own phaAB gene cluster for synthesizing 3HB-CoA, it is almost incapable of synthesizing 4HB-CoA. To address this, a heterologous gene cluster comprising ogdA, sucD, 4hbd, and orfZ has been introduced to facilitate the synthesis of 4HB-CoA. Subsequently, the endogenous phaC gene catalyzes the conversion of both 3HB-CoA and 4HB-CoA into P34HB.

Experimental characterisation

growth conditions

E. coli was cultured at 37 °C in an LB medium containing (g L−1) 10 NaCl, 10 tryptone, and 5 yeast extract. H. bluephagenesis was cultured at 37 °C in a 60-LB medium, namely, the LB medium supplemented with 60 g L−1 NaCl. A 20-LB medium indicates the LB medium containing 20 g L−1 NaCl. Moreover, 15 g L−1 agar was added before autoclaving for preparing solid media in Petric plates. Ampicillin (100 μg mL−1), chloramphenicol (25 μg mL−1), kanamycin (50 μg mL−1), or spectinomycin (100 μg mL−1) were added to the above media whenever necessary.

shake flask studies

H. bluephagenesis TD80 and its derivatives were cultivated in 50-MMF medium in shake flask studies. The 50MM medium was composed of (g/L): NaCl 50, sodium formate 15, yeast extract 1, CO(NH2)2 0.25, MgSO4 0.2, Na2HPO4·12H2O 9.65, KH2PO4 1.5, trace element solution I 10 mL/L and trace element solution II 1 mL/L. The composition of trace element solution I was (g/L): Fe(III)-NH4-citrate 5, CaCl2 2, HCl 1 M. The trace element solution II was composed of (mg/L): ZnSO4·7H2O 100, MnCl2·4H2O 30, H3BO3 300, CoCl2·6H2O 200, CuSO4·5H2O 10, NiCl2·6H2O 20 and NaMoO4·2H2O 30. The pH-value of the medium was adjusted to approximately 9.0 using 5 M NaOH.

the microbial glycerol stocks were resuscitated by streaking on fresh plates. Then constructed plasmid was transferred into Halomonas TD80 through modified conjugation method using E.coli S17-1 as donor cells[6]. Single colonies from newly-conjugated plates were picked and inoculated in the 60-LB liquid medium for 12 h at 200 rpm to acquire the first seed culture, which was further grown on a fresh 60-LB liquid medium at a volume ratio of 1%. The second seed culture was inoculated for 12 h at 200 rpm. Afterward, it was inoculated into 150-mL conical flasks containing 20 mL of the defined minimal medium at a volume ratio of 5% and cultivated for 48 h at 200 rpm. Antibiotics were added if needed. The temperature for all cultivations was 37 °C.

experimental design

Using 35g/L sodium acetate as the sole carbon source

Experimental group: Importing pSEVA321 & pSEVA341 into TD80: Compare the effects of introducing the same gene cluster into TD80 using different vectors, focusing on factors such as the 4HB molar ratio, dry weight, yield, and other relevant metrics.

Control group: Wild TD80 containing 35g/L sodium acetate as the sole carbon source: Assess the impact of introducing gene clusters

Post fermentation treatment

·Dry weight measurement: Take 15ml of bacterial solution, centrifuge evenly at 9000rpm for 5 minutes. Discard the supernatant, then add 15ml of water to resuspend the precipitate (2500rpm for 10 minutes). Centrifuge evenly for the second time, discard the supernatant, cover the tube with sealing film and puncture the hole. Place the sample in a -80 °C freezer for 2 days, then transfer it to a freeze dryer for 24 hours before weighing it.
·Content measurement: Take 30-40 mg of the sample into an esterification tube. Add 2 mL of esterification solution and 2 mL of chloroform to each tube. Heat the mixture at 99.9 °C for 4 hours, then cool it down. Add 1 mL of ultrapure water to each tube and mix well. Allow it to stand for 1 hour, then take 1 mL of the lower liquid from the filter head and analyze it using a gas chromatograph.

Data Processing and Analysis

The experimental results indicated that the 4HB molar ratio achieved by introducing the pSEVA321 was higher than that of the pSEVA341. However, the dry weight decreased somewhat compared to the wild TD80.
In order to stabilize the cell dry weight while increasing the 4HB molar ratio, we decided to explore the effect of the pSEVA321 on cell dry weight and make improvements. For further experiments, please turn to BBa_K5226075.