Team:Rock Ridge Virginia/Experiments
Protocols
Here you can find the protocols we used throughout the summer
Gene Protocols
Vaccine Platform
- This project was based on a past UVA iGEM project on minicells. We tried to use this minicell as a vaccine platform using Wolbachia Surface protein as an immunogenicity factor/adjuvant for other protein that are not very immunogenic. Our goal was to immunize animal vectors and make them immune to the Lyme bacteria. We were going to use 4 common surface proteins from Lyme: OspA, OspC, DbpA, and DbPB. We selected these proteins because based on research they are the most common protein that are expressed in the different animal vectors. Unfortunately due to the lack of resources and the need to focus, we are putting this project in our drawer for future use.
Bacteria mimic/Living vaccine project
- Idea
- After researching for the vaccine platform, we realized that the only component needed for the Lyme bacteria to colonize the Tick's gut was OspA. We started brain storming as how we could use it to combat Lyme disease. We first thought of making a bacteria that can secrete the Tick Receptor of OspA protein (TROSPA), which was shown to be active and able to bind in its soluble and truncated form to the Lyme bacteria's OspA. We quickly realized that our secreted protein may not co-localized with Borrelia burgdorferi (Lyme bacteria) and therefore be ineffective. This is how the idea of making a mimic came about, if we wanted our bacteria to co-localize with the Lyme bacteria then our bacteria would have to resemble Lyme. And the only protein needed to accomplish this is OspA. Through our research we also realized that this technique can potentially work for other insect born diseases. The only problem that we had was how to vertically transfer (parent to offspring) our mimic to the next generation of ticks, so they are also vaccinated against Lyme disease. We knew the key was the sex cells and we found our answer on another bacteria, a symbiot called Wolbachia. Wolbachia Surface Protein (WSP) is responsible for bacteria host interaction, it is the major surface protein in Wolbachia and if co-localizes well with the sex cells of female insects.
Igem parts used
BACTERIA MIMIC PROJECT ASSEMBLY OF PARTS
Strong Promoter
- Part:BBa_J23100
ttgacggctagctcagtcctaggtacagtgctagc
RBS that goes with above promoter
- BBa_B0034 Part-only sequence (12 bp)
aaagaggagaaa
Terminator
- BBa_B0014
Genes
- We used the Nucleotide Data Base from NCBI to find the OspA and WSP gene sequences.
- Translate from Expasy was used to double, triple check that we have the right protein sequence. WSP is 2151 bp, and OspA is 2475 bp.
- the sequences were then sent to Integrated DNA Technologies (IDT) and Genscrip for production.
- Harvest overnight bacterial culture by centrifuging at 6000 x g for 15 mins at 4 degrees celsius
- Resuspend the bacterial pellet in 0.3 ml of buffer P1
- Add 0.3 ml of buffer P2, mix thoroughly by vigorously inverting 4-6 times, and incubate at room temperature for 5 mins
- Add 0.3 ml pre chilled buffer P3, mix thoroughly by vigorously inverting 4-6 times, and incubate on ice for 5 mins
- centrifuge at 10,000 rpm for 10 mins. re centrifuge if the supernatant is not clear
- Equilibrate a QIAGEN-tip 20 by applying 1 ml of Buffer QBT, and allow column to empty by gravity flow
- Apply the supernatant from step 5 to the quiagen-tip and allow it to enter the resin by gravity flow
- wash the QUIAGEN-tip with 2x2 ml buffer QC. allow BUffer QC to move through the QUIAGEN-tip by gravity flow
- Elute DNA with 0.8 ml Buffer QF into a clean 2 ml vessel.
- precipitate DNA by adding 0.56 ml (0.7 volumes (70%)) room-temperature isopropanol to the eluted DNA and mix. Centrifuge at 15,000 xg for 30 mins. Carefully decant the supernatant
- Add 0.7 volumes of isopropanol and keep on ice for 10 mins
- Transfer solution into a mini-spin column and centrifuge for 1 min. at 10,000 rpm .
- Wash with buffer 500ul AW1
- Then wash with buffer 500ul AW2
- Spin dry for 1 min at 10,000rpm
- Elute DNA with 100ul ultrapure water
Digest
- Enzyme Master Mix for Plasmid Backbone (25ul total, for 5 rxns)
- Digest Plasmid Backbone
- 5 ul NEB Buffer
- 0.5 ul BSA
- 0.5 ul EcoRI-HF
- 0.5 ul PstI
- 18 ul dH20
- Add 19 ul linearized plasmid backbone (25ng/ul for 100ng total)
- Add 4 ul of Enzyme Master Mix
- Digest 37C/30 min, heat kill 80C/20 min
PCR protocols
GFP PCR
- Volume of 25 ul:
- WA2F = 3 ul
- GSV-1R = 3 ul
- GFP DNA = 2 ul
- Distilled, autoclaved water = 17 ul
- Volume of 50ul:
- WA2F = 6 ul
- GSV-1R = 6 ul
- GFP DNA = 4ul
- Distilled, autoclaved water = 34 ul
OspA PCR
- Volume of 25 ul:
- WA2F = 3 ul
- WA3R = 3 ul
- OspA DNA = 2 ul
- Distilled, autoclaved water = 17 ul
- Volume of 50 ul:
- WA2F = 6 ul
- WA3R = 6 ul
- OspA DNA = 4 ul
- Distilled, autoclaved water = 34 ul
WSP PCR
- Volume of 25 ul:
- VPW1F = 3 ul
- WA3R = 3 ul
- WSP DNA = 2 ul
- Distilled, autoclaved water = 17 ul
- Volume of 50 ul:
- VPW1F = 6ul
- WA3R = 6 ul
- WSP DNA = 4 ul
- Distilled, autoclaved water = 34 ul
Plasmid Mini Kit Protocol (modified protocol when hi speed centrifuge is not available
- After researching for the vaccine platform, we realized that the only component needed for the Lyme bacteria to colonize the Tick's gut was OspA. We started brain storming as how we could use it to combat Lyme disease. We first thought of making a bacteria that can secrete the Tick Receptor of OspA protein (TROSPA), which was shown to be active and able to bind in its soluble and truncated form to the Lyme bacteria's OspA. We quickly realized that our secreted protein may not co-localized with Borrelia burgdorferi (Lyme bacteria) and therefore be ineffective. This is how the idea of making a mimic came about, if we wanted our bacteria to co-localize with the Lyme bacteria then our bacteria would have to resemble Lyme. And the only protein needed to accomplish this is OspA. Through our research we also realized that this technique can potentially work for other insect born diseases. The only problem that we had was how to vertically transfer (parent to offspring) our mimic to the next generation of ticks, so they are also vaccinated against Lyme disease. We knew the key was the sex cells and we found our answer on another bacteria, a symbiot called Wolbachia. Wolbachia Surface Protein (WSP) is responsible for bacteria host interaction, it is the major surface protein in Wolbachia and if co-localizes well with the sex cells of female insects.