Phage Purification

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March

3/15/13

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

AC

I spent time trying to find a good procedure to propagate phage in a liquid medium. I 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. Then, 5 mL top agar was added to each culture tube. Each tube was then plated and incubated at 37 degrees C.

Amber - T2 Arick - T5 Darren - T3

DL 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 could have caused some contamination. While filling my -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.

RESULTS: 3/22 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 old.

3/22/13

AC 3/22

We had several sources of error in our procedure. First off there was contamination that affected our results. The micropipette tips that we used had not been autoclaved. This caused contamination to several plates. We also microwaved the augar instead of using a heat bath. This caused there to be tons of condensation in the plates and the augar to crack. We didn’t have any plates that had phage present.

Results: Despite the sources of error, it was pretty clear that the phage did not react with the bacteria. This was either because the phage was not viable, or because of the contamination.

AB 3/22/2013

None of the plates showed phage. A big problem that we had was contamination, but our agar was also crystal looking and cracked in several places. sources of contamination were accidental use of unautoclaved pipettte tips, and the agar was microwaved and exploded, causing water condensed in the microwave to fall into the agar. Results: No phage, lots of contamination. We may need a new source of phage, or just better lab technique.

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

DL 3/22

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

AB 3/25/2013

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.

DL 3/25

What we need to do this week: Get a list of reagents, make sure we have the procedures down.

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.

Phage Purification

From 2013.igem.org

Contents

March

3/15/13

3/18/13

3/20/13

3/22/13

3/25/13

3/27/13

3/29/13

April

4/1/13

4/4/13

4/5/13

4/8/13

4/10/13

4/12/13

4/15/13

May

5/1/13

5/3/13

5/6/13

5/8/13

5/10/13

5/13/13

5/15/13

5/17/13

5/20/13

5/22/13

5/24/13

5/27/13

5/29/13

5/31/13

June

6/3/13

6/5/13

6/7/13

6/10/13

6/12/13

6/14/13

6/17/13

6/19/13

6/21/13

6/24/13

6/26/13

6/28/13

July

7/1/13

7/3/13

7/5/13

7/8/13

7/10/13

7/12/13

7/15/13

7/17/13

7/19/13

7/22/13

7/24/13

7/26/13

7/29/13

7/31/13

August

8/2/13

8/5/13

8/7/13

8/9/13

8/12/13

8/14/13

8/16/13

8/19/13

8/21/13

8/23/13

8/26/13

8/28/13

8/30/13

September

9/2/13

9/4/13

9/6/13