Our engineering design cycle.
In the simplest conception of our project, we need to produce different types of spidroins, the basic unit of spider silk, in order to adjust the mechanical properties of spider silk by mixing various spidroins with different properties. Our aim is to engineer some recombinant E. coli to express the spidroins that we are interested in. We chose E. coli as our chassis as it is easy to handle and has a rapid growth rate compared to other chassis, such as yeast or mammalian cell lines. Also, it is easy to transform, which is a crucial point of concern in a high school setting.
In the process of creating the recombinant plasmid and expressing spidroin, we will use both the Top10 strain and the BL21 strain. Due to the nature of digest and ligate assembly, the chance of the recombinant plasmid in concern being formed is very low. To amplify and separate the required plasmid from other undesired plasmids, we will first transform the plasmids into E. coli Top10 so that they can be plated, selected and screened with colony PCR. During the process, the recombinant E. coli will divide and replicate to produce more copies of itself, as well as making copies of the desired recombinant plasmid. As E. coli Top10 is designed for copying plasmids, using this strain enables the rapid production of large amounts of plasmids in a short period of time. Moreover, it is possible to express the gene directly in this strain. This is great for test runs. To compensate for this, we will extract the desired recombinant plasmid from E. coli Top10 by miniprep, and transform E. coli BL21 with the extracted plasmids. This way, we can take advantage of the superior expression capabilities of E. coli BL21 for expressing the spidroins that we need, as well as using Top10 for plasmid amplification.
To assemble the recombinant plasmid that we want, which contains the gene of interest and other regulatory sequences required for expression, such as ribosome binding site and inducible promoter, we will use digest and ligate assembly to create the plasmid. We chose digest and ligate assembly as this is cheaper and easier to implement compared to other methods like Gibson assembly. The backbone of our choices are the pET-SUMO , pBAD-HisB and pET-22b(+) expression plasmid. The inducer of pET-SUMO and pET-22b(+) is IPTG, and the inducer of pET-HisB is arabinose. Thus, different inducers are needed. In our original plan, we plan to control the concentration of spidroins expressed by E. coli by adding different amounts of each type of inducer so as to adjust the mechanical properties of spider silk. However, after consulting the professionals, we ceased this plan and decided to adjust the properties of spider silk by mixing proportional amounts of various spirdroin after expression. pET-SUMO is low-copy plasmid, and pBAD-HisB and pET-22b(+) are high-copy plasmid. Using two different types of plasmid can adjust the efficiency of spridrion produced. We ordered the plasmid containing gene of interest from IDT, which removes the hassle of obtaining real spiders and extracting DNA from them. We will transform the plasmid into E. coli Top10 and culture. Top10 is designed for copying plasmids, so we can extract the plasmids from the cells by miniprep. Furthermore, we will digest and extract the target genes from the plasmids.
NT-2Rep-CT is a common mini-sprodroin used in many other iGEM teams. A SpyTag is added in the end and two restriction sites are added in both ends of the gene. NT-2Rep-CT-SpyTag can be functionalized with various SpyCatcher-fused proteins to bind with different proteins for different purposes.
To test the design, we will assemble it in the lab and transform it into E. coli as mentioned above. After transformation, we will culture the recombinant E. coli in LB broth and add inducers to express the spidroin. We then extract the spidroin via his-tag chromatography and check if the result is as expected or not by SDS-PAGE. If the result is as expected, we should see a band on the gel at the position corresponding to its molecular weight.
As there is only one component to be inserted into the plasmid backbone, that is, our gene of interest, we decided to use digest and ligate assembly. Due to the nature of digest and ligate assembly, scar sites will be introduced. However, it is expected that the scar sites will not interfere with the expression of the protein as translation only starts at the start codon and should end at the stop codons. Thus, the ribosome should ignore the scar sites before and after the gene of interest.
To verify the assembled plasmid actually contains the insert, we will perform colony PCR on the colonies that have grown on an antibiotic-agar plate. The plasmid extracted by miniprep will then be used for sequencing to confirm sequence integrity. We will use sanger sequencing as it is the most reliable sequencing method. The primers that will be used bind to the plasmid backbone and should be able to sequence the full insert. We also double-check by doing a restriction digest on the plasmid, and see if the digest profile matches the theoretical digest profile. Since we used two restriction sites in the assembly process, we do not have to worry about inverted inserts. To express the spidroin in concern, we have to transfer the plasmid to another chassis, namely from E. coli Top10 to E. coli BL21. To do this, we will take the plasmid and do another transformation into E. coli BL21 for expression.
To ensure the spidroin expressed is what we want, we will perform SDS-PAGE on the extracted protein. It is expected that we should see a band present according to the molecular weight of the spidroin.
To validate that the spidroins are expressed correctly, we will perform SDS-PAGE on the extracted proteins. A vertical gel electrophoresis tank will be needed. This will provide qualitative measurements of the spidroin in concern. Also, different samples would be taken in different steps of protein purification. That can let us know if the gene expresses correctly and does the protein purification work. Below shows the details.
| Samples | Treatment | Content | Predicted results |
|---|---|---|---|
| 1 | Culture solution after sonication | Lysed cell with all cell content | Lots of bands and should include the target band |
| 2 | Supernatant after centrifuge | Cell pellet and insoluble proteins are removed | Less bands than sample 1 and should include the target band |
| 3 | The flow out after adding wash buffer | Soluble protein w/o His-tag | Similar to sample 2, but doesn’t include target band |
| 4 | The flow out eluted by buffer B | Our target protein | Show target band |