Experiments

pET-SUMO

The plasmid consists of 3 parts: the origin of replication, kanamycin resistance gene and the expression system. The origin of replication is needed for the propagation of the plasmid in E. coli. The kanamycin resistance gene gives transformants resistance to kanamycin for selection purposes. The expression system consists of an inducible promoter called lacI promoter, restriction sites for enzymes SacI and SalI, the gene of interest and the T7 terminator. The inducible promoter and the terminator combined allows for the regulation of the gene of interest using IPTG as the inducer. The restriction sites are used to clone the gene of interest into the plasmid backbone. The gene of interest encodes the spidroin that we want to express. The expression system expresses the spidroin with a his-tag which helps in the isolation and purification of the desired spidroin.

pBAD-HisB

Compared with pET-sumo, the most essential differences of pBAD-HisB plasmid are inducer promoter, antibiotic resistance gene and restriction sites. L-arabinose operon is the inducer promoter instead of lacl. Thus, arabinose is used instead to allow the expression. The Ampicillin resistance gene allows the transformants to resist another antibiotic, ampicillin. The enzymes used for digestion are EcoRI and HindIII.

pET-22b(+)

This is a common plasmid with high copy plasmid. The enzymes for restriction digestion are XbaI and Xhol. The inducer used for expression is IPTG, same as pET-SUMO. The plasmid contains an ampicillin resistance gene which allows the transformants to resist ampicillin.

After modelling, we found that the minimal concentration of IPTG for protein expression is around 0.001M. In order to lower the cost of spridroin production, we will choose 0.001M for protein expression. Also, the modelling results show that proportional expression is the most suitable way to get and adjust the properties of spider silk made.

1. Transformation

Before transformation, the competent cells were prepared by mixing the cultured Top10, which is designed for copying plasmids, calcium chloride and calcium chloride with glycerol in different processes. This made the cells become positively charged so that they were easier to take up plasmids which were negatively charged. Since the competent cells were highly unstable, they were stored at -80°C before use.

Firstly, we transformed the plasmids backbones and plasmids containing target genes which were ordered from IDT into the component cells, E. coli Top10 by undergoing heat shock. After that, we recovered and plated the cells on the LB agar plate with kanamycin or ampicillin. Only the E. coli that had taken up the plasmids could grow and be selected.

Incubated the cells at 37°C for 24-28 hours in order to form colonies for further selection.

2. Liquid culture

We selected the colonies from the agar plate, put them in LB broth with proportional amounts of antibiotics to ensure only the cells which have received expected plasmids can replicate in the culture tube. Then, we put the LB broth in a shaking incubator for incubation for around 24 hours.

3. Miniprep

After culture, we pelted the cells, resuspended them with the resuspension buffer, broke the cells with the lysis buffer and neutralised the solution with the neutralisation buffer.

Then, we added the binding buffer and transferred the solution into spin miniprep kits, centrifuged the kits. The plasmids were in the column now. Moreover, we washed away the unwanted materials with the wash buffer twice. Finally, we extracted the plasmid by adding the elution buffer and centrifuging them.

Final prducts

• large amount of plasmids containing target genes
• large amount of plasmid backbones

1.Digestion

Firstly, we digested the plasmids containing target genes with corresponding restriction enzymes. Sall and Sacl for pET-SUMO plasmid, HindIII and EcoRI for pBAD-HisB plasmid and XbaI and Xhol for pET-22b(+).

After digestion, we ran gel to ensure the target genes were digested, two bands should be formed, and we cut out the band with lower size which matches the band size of the genes coding spidroin from the agarose gel. Then, we carried out the gene clean, dissolved the gel with the binding buffer, washed away the impurities with the washing buffer and extracted the genes with the elution buffer.

The next step was digesting the plasmid backbones with the same enzymes we mentioned. As a result, both inserts and backbones were digested to create complementary sticky ends for ligation.

2. Ligation

The complementary sticky ends on the backbone and inserts were ligated to create the desired recombinant plasmid.

3. Transformation

In this process, we used the same method we mentioned before. Recombinant plasmid would be transformed into E. coli Top10.

After that, recovered and plated the cells on the LB agar plate with kanamycin or ampicillin. Only the E. coli that had taken up the plasmids could grow and be selected.

Incubated the cells at 37°C for 24-28 hours in order to form colonies for further selection.

4. Colony PCR

After incubation, the colonies were picked to perform colony PCR in order to distinguish between undigested plasmid transformants and recombinant plasmids transformants.

The colonies with the correct band size were extracted from the plate and cultured by liquid culture in order to get a large amount of plasmids for further analysis.

5. Gel electrophoresis and restriction digestion

In order to check the recombinant plasmid, we digested the plasmids with corresponding restriction enzymes and carried out gel electrophoresis. If the insert and plasmid ligated successfully, two bands should be formed and the band size should match the size of backbones and the target gene.

6. Sanger sequence

To verify the recombinant plasmid actually contains the anticipated inserts, we sent the plasmids to the companies to check the sequence of the recombinant plasmids.

1.Transformation

In this process, we used the same method we mentioned before. However, we should transform the recombinant plasmid into E. coli BL21 this time.

After that, recovered and plated the cells on the LB agar plate with kanamycin or ampicillin. Only the E. coli that had taken up the plasmids could grow and be selected.

Incubated the cells at 37°C for 24-28 hours in order to form colonies for further selection.

2. Liquid culture

Firstly, we prepared seed culture because freshy culture is recommended for large culture and thus inducer incubation. We selected one of the colonies from the agar plate and cultured by LB both, corresponding antibiotics were added to ensure only the cells which received the plasmids to grow and underwent cell division. We inoculated the E. coli overnight.

The next morning, a certain amount of seed culture solution was transferred into a large amount of LB (5ml seed culture solution : 1L LB culture solution) at 37°C until the OD600 reached 0.8. (It should be around 4 hrs for BL21.)

IPTG incubation

After the OD600 reached 0.8, we induced the protein expression with a corresponding amount of inducers. The LB culture would be incubated with 0.001M IPTG/arabinose in room temperature for around 12hrs.

Protein purification

After IPTG/arabinose incubation, we harvested the cells by centrifugation.Then we discarded the supernatant, resuspended the cell pellet with bufferA (300mM NaCl, 50mM Tris, pH8.0), and added 1mM Phenylmethylsulphonyl fluoride (PMSF). Moreover, we lysed the cells by sonication on ice and collected the supernatant by centrifugation. Finally, we loaded the collected supernatant onto Ni-NTA column, used 5% bufferB (300mM NaCl, 50mM Tris, 250mM imidazole, pH 8.0) and 95% buffer A to wash out unwanted protein at least twice, and use 100% buffer B to elute target protein.

SDS-PAGE

To verify the protein extracted was what we wanted, we had to perform the SDS-PAGE to check the length of the proteins. To ensure the protein detected was not contaminated, we also ran negative samples.

Protein concentration

After elution, we filtered the eluted protein solution through the 0.45μm syringe filter to remove insoluble impurities. Then we measured the concentration of the protein which has size around 35kDA using Nanodrop. After that, we transferred the solution into centrifugal concentrators with 10000 MWCO, centrifuged the solution and discarded the filtrate. And then, we measured the concentration of the protein solution. We repeated the whole process until the protein concentration reached at least 200 mg/ml.

Silk Fabrication

The concentrated protein is transferred into a syringe and stood overnight to ensure no gas bubbles in the solution. The next day, the spider silk was ejected from the syringe into the mixture of NaAc and NaCl with pH5.0 using a syringe pump. The spider silk should be immersed in a buffer for at least two days to ensure it is fully solidified.