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

Overview

March-April

May-June

July-August

September-October

We will take a 500 flask; with 50 mL LB broth, and place a culture from the W3310 E.coli culture plate to grow overnight in the 37 C incubator

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

4/4/13

- We made glycerol stocks W3110 E. Coli

We put .5mL glycerol in cryovials and .5mL liquid culture from the overnights prepared yesterday.

We vortexed each tube for 10 seconds and then we stored the tubes in the -80°C freezer in Dr. Grose’s lab.

4/5/13

-We did spot tests of phage on E. Coli BL21

We plated together .5mL of BL21 with 1XLBTA.

We separated the plates into different segments and then spot tested the following phages.

14 T3

13 T5

10 T2

20OX171

40ØX174

The plates were then put in a 37°C incubator

-Results:

There were no plaques on any of the plates.

There was no contamination in the control quadrant.

A uniform bacterial lawn formed with no phage infection.

4/8/13

-We performed a phage titer test to determine which bacteria would be most viable for our phage propagation.

We used BL21 and W3110 strains of E. Coli.

We placed 100 µL of broth into 5 ependorf tubes.

We used as phage 10 µL of 1L, 10L, 40T4, T4DOS, and T4 infected phage and placed them each in one of tubes labeled -1.

We performed a dilution series taking out 10 µL each time and placing it into the next tube, 5 times. The total volume in the last tube was 110 µL.

We added 4.5 mL of 1X top agar to .5 mL of broth and plated it on LB plates.

We spotted each concentration on the plates and incubated it overnight at 37°C.

-Results from 04/08:

Phage 10L w/ w3110 had large scale infection every concentration

Phage 10L w/ BL21 had infection in very large concentrations

Phage 1L w/ w3110 had infection in very large concentrations

Phage 1L w/ BL21 had infection in very large concentrations

Phage T4 DOS w/ w3110 had infection in very large concentrations

Phage T4 DOS w/ BL21 had infection in very large concentrations

Phage 40 T4 w/ w311o had no phage infection

Phage 40 T4 w/ BL21 had large scale infection at every concentration

Phage T4 inf w/ w311o had large scale infection at every concentration

Phage T4 inf w/ BL21 had large scale infection at every concentration

We had a significant amount of running that occurred on almost every plate, so the results were a little difficult to read. We concluded that the phage is a high enough concentration that we would have to do another phage titer to a smaller dilution in order to determine actual concentration. The controls were all a lawn of bacteria.

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