Team:UCSC/Design
Biological Design Challenges
A. Determine the optimal concentration of IDP to protect the NDV vaccine.
B. Optimize protein production by characterizing the growth of our BL21 Xjb(DE3) Autolysis E. coli cells.
C. Determine the best method for vitrifying our IDPs.
D. Determine the highest temperature our IDPs can protect the NDV vaccine.
E. Determine which IDPs work best at protecting the NDV vaccine.
Proposal
Thermostable Vaccines
Our project aims on making a thermostable vaccine formulation by capitalizing on the protective ability of IDPs. In order to produce the IDPs, we re-designed the plasmids, expressed the proteins through E.coli, and purified them via heat solubility. Plaque assays and hemagglutination assays will be our testing methods to verify the thermostability of the formulation.
Thermostable Vaccine Formulation
A. Phase 1-Protein Production
We recieved FLAG-tagged plasmids from Pamela Silver’s Lab and non-tagged plasmids from Pielak’s Lab. pET 28b has a kanamycin resistance gene, Lac I gene and a Lac operator which helps us select for transformed E. coli and makes expression of our IDPs inducible by IPTG.
I. Plasmid Re-design
We used the online tool-NEBaseChanger to design primers for Site-Directed Mutagenesis. We changed all our plasmids by substituting the N-terminus FLAG-tag with a 6x His-tag. Our Site-Directed Mutagenesis protocol was designed by referring to the Q5 Hot Start protocol from NewEngland Biolabs. The thermocycling conditions of the PCR was designed for touchdown PCR. We checked our product by sequencing.
Figure 1. Sequence of the FLAG-tag from the original plasmid we received. Highlighted part was substituted with His-tag.
Figure 2. Substitution primer for site-directed mutagenesis from NEBaseChanger.
II. Protein expression and Purification
We modified our protein expression and purification methods from Boothby et al. and Piszkiewicz et al. methods. The plasmids we used were E. coli codon optimized by Dr. Rodger Chang and contained a Lac I gene and a Lac operator. Constitutive expression of Lac I repressor blocks transcription unless IPTG is added to the liquid culture. 1mM IPTG is added at the mid-log phase of E. coli growth to maximize overexpression of protein. We stopped cell growth at 4hr and purified the protein by heat solubility.
B. Phase 2 Thermostability Testing
I. Diafiltration of an NDV and IDP mixture
We used a live Newcastle Disease vaccine as our model We are using the vaccine to propagate the virus in SPF chicken eggs .. Then we will place the virus in solution with varying concentrations of IDPs to form a glass-like structure by diafiltration.
II. Heat Shock
The solution of virus+IDP will be subjected to static temperatures, in increments of 10 °C from 20 °C to 60 °C. At each temperature increment, the solution will be exposed to heat for 10 mins, 30 mins, and for 60 mins.
III. Plaque and Hemagglutination Assays
After we heat shock the mixture of the NDV and IDP, we use them as the virus stock to run the plaque assays and hemagglutination assays. Plaque assays help us measure the number of infectious virus in the mixture by observation of plaque forming units. Plaque forming units are locations of the cell culture monolayer that have been infected and lysed by virus.and Hhemagglutination assays help us determine the relative concentration of virus in the stock by observation of agglutinated red blood cells. Agglutinated of red blood cells is caused by the hemagglutinin on virus binding to salic acid containing particles.
References
1.) Piszkiewicz, S., Gunn, K.H., Warmuth, O., Propst, A., Mehta, A., Nguyen, K.H., Kuhlman, E., Guseman, A.J. Stadmiller, S.S., Boothby, T.C., et al. (2019). Protecting activity of desiccated enzymes. Protein Science 28, 941–951.
2.) Boothby, T.C., Tapia, H., Brozena, A.H., Piszkiewicz, S., Smith, A.E., Giovannini, I., Rebecchi, L., Pielak, G.J., Koshland, D., and Goldstein, B. (2017). Tardigrades Use Intrinsically Disordered Proteins to Survive Desiccation. Molecular Cell 65, 975-984.e5.
