Team:BYU Provo/Notebook/Phage Purification/Winterexp/Period3/Dailylog

Overview

March-April

May-June

July-August

September-October

We put a colony from our W3110 plate of E.coli into 1mL LB broth and placed in the 37 C incubator overnight. We did this with 5 colonies.

-Results: the control tube of broth showed no contamination.

-We also put 500microL of 40% glycerol into cryovials for use tomorrow to prepare freezer sotcks.

4/3/13

- Today we will be setting up an overnight culture of E. coli, to grow for use with phage amplification.

We will take a 500 flask; with 50 mL LB broth, and place a culture form the E.coli culture plate to grow overnight.

We will create a glycerol stock of the E.coli culture to save in the -80 C freezer.

Liquid culture 4/2/2013

We put a colony from our W3110 plate of E.coli into 1mL LB broth and placed in the 37 C incubator overnight. We did this with 5 colonies.

Results: the control tube of broth showed no contamination. We also put 500microL of 40% glycerol into cryovials for use tomorrow to prepare freezer sotcks.

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?

-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

- 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.

3/27/13

AC 3/27/2013

Found a great article on the structure and proteins of bacteriophage T4 http://www.farisaka.bio.titech.ac.jp/text/CMLS-Leiman-20031.pdf The paper goes into detail on the morphogenesis of the capsid head, tail, and LTF. I will be using this paper for the bulk of my presentation on Monday. The main proteins of my focus will be:

GP-23

160 hexamers

GP-24

11 pentamers

GP-20

dodecamer portal vertex

GP-SOC & GP-HOC

increased vitality

T = 13 & Q = 21

DL 3/27

Capsid formation can follow several different pathways. This first is which proteins come together by single bonds, gradually forming the different parts that make up the capsid. In the second pathway, different parts are formed and then come together making multiple bonds at one time.

T4 structure http://link.springer.com/content/pdf/10.1007%2Fs00018-003-3072-1 shell has icosohedral ends and a cylindrical equatorial midsection that has a unique portal vertex where the tail attaches

head has gp hoc and gp soc attached to the outside of it gp soc helps maintain head integrity in extreme environments but neither gp hoc or gp soc are required head and midsection are formed by the gene product (gp)23

AB 3/27/2013

How they use T7 in medicine - kit from Novagen - http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3499470/ Bacteriophage T7 has six tail fibers that attatch to E.coli. These fibers are homotrimers (composed of three identical units of polypeptide) of gene 17. These kinked fibers attach to the LPS on E.coli.

Replication: The bacteriophage T7 replisome is an ideal model system for studying replication dynamics because it recapitulates the key features of more complicated systems, yet can be reconstituted in vitro with only four proteins. DNA synthesis is carried out by a stable, one-to-one complex of gene 5 protein (gp5) and thioredoxin, a processivity factor produced by the E. coli host. Gene 4 protein (gp4) contains both helicase and primase domains whereas gene 2.5 protein (gp2.5) is the single-stranded DNA binding protein (15).

Head made of 415 copies of 10B protein

Large-scale Amplification and Purification of T7 Phage Nanoparticles

The T7-p66, T7-p66x2 and T7-wt nanoparticles were propagated in the log phase culture of E. coli BL21 (OD600~0.8) grown in M9-LB broth (LB broth supplemented with 50 ml 20X M9 salts, 20 ml 20% glucose and 1 ml of 1 M MgSO4 per liter) at a multiplicity of infection (MOI) of 0.001 and incubated at 37°C until complete lysis of the culture (3–6 hours). Thirty minutes before removing the culture from the shaker, DNAse I and RNAse A (Roche, Germany) were added to degrade released bacterial nucleic acids. T7 phage nanoparticles were precipitated from the culture supernatant by addition of 1 M NaCl and 10% polyethylene glycol (PEG 6000, Merck) followed by overnight incubation at 4°C. The T7 phage pellet was resuspended in Tris-NaCl buffer (PH 8) and PEG and cell debris was removed by centrifugation at 10,000 rpm for 10 min. To remove residual PEG and debris, an equal volume of chloroform was added, gently inverted and the top aqueous phase was harvested after low-speed centrifugation at 4°C. The purified T7 nanoparticles were sterilized using a pyrogen-free 0.2 µm pore-size cellulose acetate filter (Millipore) and stored at 4°C until further analysis.

Removal of Bacterial Endotoxin from T7 Phage Nanoparticles

The bacterial endotoxin (LPS) concentration in all T7 nanoparticle preparations was determined in triplicate using a sensitive colorimetric Limulus Amebocyte Lysate (LAL) QCL-1000® kit (Lonza, USA) according to the manufacturer's instructions. LPS was removed from T7 phage nanoparticles based on a method for removal of endotoxin from protein solutions by phase separation using Triton X-114 as described by Aida et al. [22] and modified by Hashemi et al. (manuscript submitted for publication).

Genes of T7: http://www.uniprot.org/uniprot/?query=organism%3a10760+keyword%3a1185&offset=25 Thus phage T7 has an icosahedral shape with an edge of 37.7±0.5nm, a volume of (120±10)x103nm3, and a small tail that is 6-7% of the head volume. (STRUCTURE OF BACTERIOPHAGE T7 Small-AngleX-rayandNeutronScatteringStudy)

How T7 infects: http://www.sciencemag.org/content/339/6119/576.full.pdf?sid=178e4a68-d44f-4f10-b403-1ef7f893efa8 (excellent pictures for structure)

3/29/13

DL 3/29 We prepared our presentation on a power point for this upcoming Monday. We will be presenting on the background and structure of T7 and T4

AC 3/29

We put together our powerpoint presentation for Monday. I took all my information from http://www.farisaka.bio.titech.ac.jp/text/CMLS-Leiman-20031.pdf