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3/15/13
- TODAY MARKS THE START OF THE PHAGE PURIFICATION TEAM!
- Today we began researching for a procedure to begin purifying phage. We have found that T7 can self assemble with scaffolding proteins without forming procapsids, and that T4 has only been know to form procapsids. We may not have to worry about T4 if the other group isn't using it.
- Important findings
- Phage have been purified before
- Phage capsids can self assemble
- Phage (amber strain) can have their genes knocked out to make a hollow phage
3/18/13
- Priorities List:
- Find out how to put drugs into the capsid
- Possibly contact F.W. Studier for his amber t7 phage strain
- Next class we plan on growing up phage so that we will have a decent amount of phage to work with. We have several procedures that we plan on testing so that we can see if we can purify the protein capsid. We hope to be proceeding with these as soon as we have phage that we can use.
- Another issue that we need to consider is how to get drugs into the capsid.
- We need to be able to test and see if we can actually fill our empty capsids with a material.
- We have several procedures found in other papers that could possibly help us with this.
3/20/13
- We spent time trying to find a good procedure to propagate phage in a liquid medium. We finally found one that I think will work well for us.
- Today we learned a procedure for how to count phage.
- We performed a phage titer on the T4 phage to see if we have a high enough concentration to work with. This is the general procedure:
- Phage titer: reported in Pfu/mL (Pfu stands for plaque forming unit)
- mix E. coli (500 microL) with 50 microL of phage lysate
- incubate for 20 minutes
- mix with 4 mL top agar and then plate
- holes will appear in the agar that are called plaques
- How we followed it:
- We filled five test tubes full of 90 microliters of Liquid Broth each.
- We added 10 microliters of our desired phage to the first test tube and then mixed
- We took 10 microliters of the first tubes mixed solution and added it to tube 2, and followed the same procedure for each test tube down the line to tube five.
- We then labeled 6 culture tubes 0 to -5.
- In the first culture tube, we added 20 microL phage to .5 mL bacteria.
- In tubes -1 to -5 we took 20 microL from eppendorfs and added to .5 mL bacteria.
- We then allowed a 20 minute waiting period for the virus to infect the E. coli.
- 5 mL top agar was added to each culture tube.
- Each tube was then plated and incubated at 37 degrees C.
-The following teammates were assigned phage as follows:
- Amber - T2
- Arick - T5
- Darren - T3
-Results:
- We ran into several problems while doing the titer. After we had completed the titer, we found out that the pipet tips we had used were contaminated. When preparing the top agar, we had to melt it in the microwave which caused it to boil over. This also caused a lot of condensation in the plates and caused the auger to crack. This could have caused some contamination. While filling our -5 plate with top agar, there was only enough to put in 4mL of agar instead of the 5mL that was called for in our procedure.
- None of the plates had any phage. There was just a lawn of bacteria growing. This could either be because of the problems mentioned above or because the source of T3 was bad. Seeing as nobody else was able to grow any phage we believe that the source was either old or we need a new lab technique.
3/22/13
- Next step: find procedures for purifying different phage. Become experts on phage structure. possibly help with designing point mutations.
- osmotic procedure
- procedure from Dr. Grose
-Phage structure:
- T7 has proteins 10 A and 10 B for capsid, and an assembly protein
- T4 has SOC and HOC proteins
- We are currently waiting for our phage to come in so that we can begin running tests to purify the capsid. We have several procedures in mind. We need to prepare the reagents so that when the virus comes in we can immediately begin attempting to purify it. Most of the procedures we have found apply specifically to T7 but we will also try them on T4. We will continue to look for other procedures that may apply specifically to T4.
3/25/13
-Come up with a list of needed reagents for phage purification, so that when phage arrive we can begin testing procedure effectiveness for phage purification. Our findings may also be useful for the cholera group, since they also need phage to disrupt biofilms/kill cholera.
-List of materials for Phage Purification Procedure from Dr. Grose
- 1. Phage suspension buffer also called TM buffer (Tris-Mg2+ Buffer) 10 mM Tris–HCl (pH 7.2–7.5), 100 mM NaCl, 10 mM MgCl2. Addition of 1–10 mM CaCl2 in the suspension buffer may be required for the stability of some phages.
- 2. DNase I and RNase A from Bovine Pancreas (Roche or Cal- biochem). Stock solutions 1 mg/mL are stored at −20 ◦C.
- 3. Chloroform.
- 4. Sodium chloride powder: NaCl ≥ 99. 5 % for molecular biology.
- 5. Polyethylene glycol powder: PEG 6,000 (MW 5,000–7,000 g/mol) for molecular biology and biochemicalpurposes.
- 6. Cesium chloride: CsCl ≥ 99. 9 % for density gradient purification.
- 7. Ultracentrifuge equipments: Beckman L8-55M or equiva- lent.
- 8. Swinging-bucket rotors (Beckman): SW41 or SW28 and SW50 or SW65.
- 9. Centrifuge tubes (Beckman): thinwall or thickwall open-top polyallomer tubes:
- 13.2 mL, 14 mm × 89 mm for the SW41 rotor.
- 38.5 mL, 25 mm × 89 mm for the SW28 rotor.
- 5.0 mL, 13 mm × 51 mm for the SW50 or SW65 rotors.
- 10. Syringes and 18–22 gauge hypodermic needles.
- 11. Dialysis tubing: Spectra/Por molecular-porous membranetubing, MWCO 12–14,000.
- 12. Refractometer (optional).
-Another Procedure to use: https://cpt.tamu.edu/wp-content/uploads/2011/12/CsCl-phage-prep-08-17-2011.pdf
I think it uses the same materials as above, but the procedure is really easy to follow.
-Procedure for the self assembly of T13 phage - not sure if we'll need this
https://cpt.tamu.edu/wp-content/uploads/2011/12/CsCl-phage-prep-08-17-2011.pdf
Next, need to figure out what we are going to do with our purified phage - cleave tails? mutate for cholera group?
AC 03/25/2013
Plan of attack for the week: We will be coming up with a list of reagents needed for the purification of proteins. We are still waiting for purified phage to start propagating them and purifying them. In the meantime we are still reading papers on capsid structure and design as well as applications of capsids in nanotechnology. We have found some interesting articles on capsid batteries and drug epitopes. Articles are on the learningsuite website.
-Osmotic shock materials: phage, 3M Na2SO4, 2.8M MgSO4, DNAse, centrifuge that can control temperature at 10 degrees C, saline
- 1. 2.9 ml. of the above phage + 6 ml. 3M Na2SO4 for 2 min., then + 140 ml. cold water rapidly with agitation
Residual infectivity by plaque count was 5 X 108/ml.
- 2. Number 1 + 0.15 ml. saturated MgSO4 (2.8M) + 0.15 mg. DNAse left at 5°C. overnight
- 3. Number 2 centrifuged at 3500 for one half hr.; supernatant
- 4. Number 3 centrifuged at 100,000 X g for 1 hr. in a Spinco refrigerated centrifuge at 10°C.
- 5. Supernatant from number 4
- 6. Residue from number 4 dissolved in cold saline
- 7. Number 6 centrifuged at 2,000 for 15 min.; supenatant
- 8. Number 7 centrifuged at 18,000 × g in a Servall SS-2 for 1 hr.
- 9. Supernatant from number 8.
- 10. Residue from number 8 dissolved in saline
3/29/13
- Worked on our first team presentation.
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