Difference between revisions of "Part:BBa K3141000"

Revision as of 03:56, 16 October 2019

spacer; increasing proinsulin secretion

Based on others' previous research, we synthesized and modified the spacer sequence to make it more compatible to our project. Spacer BBa_K3141000, a short peptide consisting of 7 amino acids, has been proved that it is able to significantly increase proinsulin expression in Bacillus subtilis and Pichia pastoris through quantitative characterization. When using this spacer, the gene of this spacer is introduced into the N-terminal of HPI gene;then the peptide is synthesized.

In order to obtain higher proinsulin secretion, we have conducted the optimization of gene expression and insulin secretion using the yeast strain GS115 with pPICZαA plasmid as the expression vector. A interval peptide was introduced between the secretory signal peptides and proinsulin to improve gene expression for the proinsulin secretion.

[Here should be a pic]

Figure 1. plasmid map

We have selected the restriction sites as follows:
at the 5' end: EcoR I GAATTC
at the 3' end: Kpn I GGTACC

Experimental Method

I procedure:

1.Construction of vectors (extraction of plasmids, digestion, conjugation and transformation, etc.)

2.Transformation and Screening of Yeast GS115

Formula of growth media:

A.YPG medium: 2.0% tryptone, 1% yeast extract, 2% glycerol (solid medium with 1.5% agar).

B.Complete YPD medium : 2.0% tryptone, 1% yeast extract, 2% glucose.

C.Induction BMGY medium: 2.0% tryptone, 1% yeast extract, 1.34% YNB, 2% glycerol, 10mmol/L potassium phosphate (pH 6.0), biotin 4×10-5% (by aseptic filtration).

D.Induction BMMY medium : 2.0% tryptone, 1% yeast extract, 1.34% YNB, 0.5% methanol, 10mmol/L potassium phosphate (pH 6.0), biotin 4×10-5% (by sterile filtration)

II Preparation of competent Pichia pastoris cells:

Single colony in the plate bearing Pichia pastoris GS115 was picked up and inoculated into 50 mL shaker’s flask containing 5 mL YPD medium. It was incubated overnight at 30°C and 200 r/min. It was then used as a seed to inoculate 100 mL YPD liquid in 500 mL shaker’s flask to grow at 30°C and 200 r/min till OD600 = 1.2-2.0. The P. pastoris growth liquid was centrifuged at 4°C for 5 minutes at 1600×g, and the supernatant was completely removed. The pellet was re-suspended with 10 mL D-Sorbitol (conc. 1mol/L). The suspension was centrifuged at 4°C for 5 min at 1600×g. The supernatant was removed. It was repeated for 5 times. Use 1000μL 1mol/L D-Sorbitol to make final suspension, put it into ice bath.

III Electroporation of Pichia pastoris

Put 80 μL competent Pichia pastoris cell suspension into a pre-cooled 0.2cm electroporation cup, add 20 μL linearized plasmid and mix it. The mixture was placed on ice for 5 minutes (or - 20°C for 1 minute). Put the cup into the electroporator for electroporation (voltage 2.0 kV, electric shock time 5 ms). Then immediately add 1 mL pre-cooled sterile 1 mol/L D-Sorbitol and rest for 60 minutes. The MD plate was coated with 200 μL competent Pichia pastoris cell suspension and placed in an oven at 30°C for 20-60 mins. The plate is cultured for 3-5 days.

IV Screening of multi-copy Pichia pastoris recombinants

P.pastoris recombinants were randomly selected from the YPG-Zeocin (Bleomycin, a kind of antibiotic) plate with aseptic toothpicks. The recombinants were grown on the YPG-Zeocin plates with Zeocin concentration gradient (300 ug/mL, 600 ug/mL). The incubator was set at 30°C for 3-5 days. Colonies in different plates were examined daily. According to the quantitative dependence between the copy number of heterologous genes and the resistance of zeocin, it can be inferred that the cells grown only on the plates with low zeocin concentration are with low copy numbers, and the cells grown on the plate with high zeocin concentration are with high copy numbers.

VI Induced expression of Pichia pastoris recombinants

Recombinant colonies on YPD plates with different copy numbers were selected (100, 300, and 600 ug/mL Zeocin, respectively). They were used to inoculate 100 mL BMGY medium in 1000 mL flasks, and put in a shaker for 16-24 h at 30°C. When the OD600 was 2-6, the culture was replaced with same volume BMMY medium for induction under aseptic condition. The culture was continued at 28°C for 120 hours. Methanol was added every 24 hours to make total concentration of methanol in the liquid to be 0.5%. After fermentation, the supernatant was precipitated with trichloroacetic acid (TCA). 50% TCA was added with 0.2% sodium deoxycholate. 25% TCA was mixed with 75% medium centrifugal supernatant (volume ratio) evenly and placed on ice for 30 min. It was then centrifuged (4℃, 20,000 xg, for 20 mins), the supernatant was removed, pellets were resuspended with 10% TCA. It was centrifuged again (the same condition). Remove the supernatant, suspend it with cold acetone (- 20℃) and centrifuge it (the same condition). Precipitation was dried by a blower and electrophoretic detection was carried out by SDS buffer solution.

Experiment Results

1.Screening of Pichia pastoris transformants:

[HERE SHOULD BE A FIGURE]

Figure 2. Gradient screening of the transformants.
A: 300 ug/mL Zeocin Screening
B:600 ug/mL Zeocin Screening

It can be seen from Figure 2 that some Pichia pastoris may have been integrated with multiple copies of gene expression nuclei, they may grow faster, have larger colony diameter, and may express insulin genes more efficiently.

2. 8 high-copy transformants were selected and cultured in shaking flask. Their insulin expression levels were detected. The results of protein electrophoresis were as follows:

[HERE SHOULD BE A FIGURE]

Figure 3. Electrophoresis for insulin expression

It is found that the highest expression level of recombinant protein can reach 150 mg/L, accounting for about 40% of total secreted protein for our project (with novel insertion of an interval peptide). In comparison, the highest secretory expression of proinsulin was only 32 mg/L in Pichia pastoris transformants reported in the literature. The expression of proinsulin was increased by about 5 times after our synthetic biology work to modify the N-terminal of the proinsulin.

Sequence and Features