Team:BYU Provo/Notebook/SmallPhage/Winterexp/Period1/Dailylog

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Small Phage March - April Notebook: March 15 - March 31 Daily Log

3/15/13

-Today we began research on phage purification -Focused on T4 and T7 as this is what the other groups will be focusing on

-T7 can self assemble with the help of scaffolding proteins without forming procapsids

-T4 assembles using procapsids

-Some phage can have their DNA polymerase genes knocked out to make a hollow phage

-Possibility for making ghost capsids

3/16/13

- Created a priority list of things we need to complete:

-Find out how drugs are put into capsids

-Possibly contact F. W. Studier for his amber T7 phage strain

-This strain had the knocked out DNA polymerase gene

-Perform a phage titer on the phage we have to see if we have enough to work with

-Find a procedure that we can use to test if our purified phage are viable by filling the capsid with liquid

3/18/13

- Presented current understanding and plans for the future

Decided on a two-focus attack

Evolution – selecting naturally occurring smaller phages

Site directed mutagenesis – using genome info and comparative studies to identify sites to target

- More background research

Possible ways of making phage smaller

3/20/13

- Background research on phiX174: the only phage we have in stock

- Performed 3.20 Phage Viability Test

3/21/13

- Checked up on results for 3.20 Phage Viability Test

3/22/13

- Discussed results from tittering experiment (preliminary experiment 1)

Contamination mostly likely resulted from the step involving suspending phage in LB – obvious contamination in the LB bottle

One of the stock phage solution had contamination as well

- Discussed step of attack with Dr. Grose

Decided to go with T7, if necessary Qbeta

Need to learn to make top agar at various concentrations

Need to do more background research and decide whether to assemble phage capsid in vitro (plasmid + E coli) or do direct mutagenesis of phage genome

Need to correspond with the isolation team

- Sequencing will be for individual genes to cut down cost

Need to design primers and get to know the genome of the phage

- Learnt about Mega5 to compare genome and protein sequence

3/25/13

- Reported on past week and plans for this week

From last week: titering experiment

This week

Learn to make top agar at various concentrations

Background research to determine in vitro assembly vs altering genome – look into specific techniques

Comparing genome of phage and decide on possible site-directed mutagenesis options

- Start working on designing our site directed mutagenesis

Qbeta vs MS2

Look for places where sequences are significantly different

Might be worthwhile to look at capsid structure to identify the regions where interactions between subunits take place

Qbeta vs T7 major

No real consensus – more worthwhile to compare capsid protein sequence of T7 with those that have similar size to it

T7 major vs minor

Minor is longer, but not necessary – the tail overhang is due to ribosome moving two codons downstream instead of three

Suggest we can direct mutation to the poly-U site and prevent ribosome slippage

Qbeta major vs minor

Just continue transcribing after it reaches the stop codon. What does the stop codon code for?

- Research into in-vitro assembly vs direct mutation of phage genome

It seems that we’ll need to clone the genome of the phage into a plasmid and let it assemble in an E coli

Using chemicals we can induce random mutations in phage – might be worthwhile if selection in agar is not working as well.

3/27/13

- More research on genome of enterobacteria phage

Generation of the major and minor capsid in Q beta

Capsid protein information research

Capsid protein sequence comparison

- Outlined protocol for producing stock top agar

4.3 Top Agar Stock Preparation

3/29/13

- Worked on our first team presentation.

@@ Line 4: / Line 4: @@
 {| width="100%"
-| colspan="3" | <font color="#333399" size="5" font face="Calibri"> '''Phage Purification March - April Notebook: March 15 - March 31 Daily Log'''</font>
+| colspan="3" | <font color="#333399" size="5" font face="Calibri"> '''Small Phage March - April Notebook: March 15 - March 31 Daily Log'''</font>
 <br>
@@ Line 14: / Line 14: @@
 <font color="#333399" size="3" font face="Calibri">
-: [[Team:BYU_Provo/Phage_Purification|Overview]]
+: [[Team:BYU_Provo/Small_Phage|Overview]]
-: [[Team:BYU Provo/Notebook/Phage_Purification/Winterexp|March-April]]
+: [[Team:BYU Provo/Notebook/SmallPhage/Winterexp|March-April]]
-: [[Team:BYU Provo/Notebook/Phage_Purification/Springexp|May-June]]
+: [[Team:BYU Provo/Notebook/SmallPhage/Springexp|May-June]]
-: [[Team:BYU Provo/Notebook/Phage_Purification/Summerexp|July-August]]
+: [[Team:BYU Provo/Notebook/SmallPhage/Summerexp|July-August]]
-: [[Team:BYU Provo/Notebook/Phage_Purification/Fallexp|September-October]]
+: [[Team:BYU Provo/Notebook/SmallPhage/Fallexp|September-October]]
 </font>
@@ Line 32: / Line 32: @@
 <font size="4"> '''3/15/13''' </font>
-- TODAY MARKS THE START OF THE PHAGE PURIFICATION TEAM!
+-Today we began research on phage purification
+-Focused on T4 and T7 as this is what the other groups will be focusing on
+:-T7 can self assemble with the help of scaffolding proteins without forming procapsids
+:-T4 assembles using procapsids
+-Some phage can have their DNA polymerase genes knocked out to make a hollow phage
+:-Possibility for making ghost capsids
+<br>
-- 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.
+<font size="4"> '''3/16/13''' </font>
-- Important findings
+- Created a priority list of things we need to complete:
-:Phage have been purified before
+:-Find out how drugs are put into capsids
-:Phage capsids can self assemble
+:-Possibly contact F. W. Studier for his amber T7 phage strain
-:Phage (amber strain) can have their genes knocked out to make a hollow phage
+::-This strain had the knocked out DNA polymerase gene
+:-Perform a phage titer on the phage we have to see if we have enough to work with
+:-Find a procedure that we can use to test if our purified phage are viable by filling the capsid with liquid
 <br>
@@ Line 45: / Line 53: @@
 <font size="4"> '''3/18/13''' </font>
-- Priorities List:
+- Presented current understanding and plans for the future
-:Find out how to put drugs into the capsid
+: Decided on a two-focus attack
-:Possibly contact F.W. Studier for his amber t7 phage strain
+:: Evolution – selecting naturally occurring smaller phages
-- 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.
+:: Site directed mutagenesis – using genome info and comparative studies to identify sites to target
-- Another issue that we need to consider is how to get drugs into the capsid.
+- More background research
-:We need to be able to test and see if we can actually fill our empty capsids with a material.
+: Possible ways of making phage smaller
-:We have several procedures found in other papers that could possibly help us with this.
 <br>
@@ Line 57: / Line 64: @@
 <font size="4"> '''3/20/13''' </font>
-- 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.
+- Background research on phiX174: the only phage we have in stock
-- Today we learned a procedure for how to count phage.
+- Performed [[Team:BYU_Provo/Notebook/SmallPhage/Winterexp/Period1/Exp/3.20 Phage Viability Test|3.20 Phage Viability Test]]
-- 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:
+<br>
-: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:
+<font size="4"> '''3/21/13''' </font>
-: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.
+- Checked up on results for [[Team:BYU_Provo/Notebook/SmallPhage/Winterexp/Period1/Exp/3.20 Phage Viability Test|3.20 Phage Viability Test]]
 <br>
@@ Line 92: / Line 78: @@
 <font size="4"> '''3/22/13''' </font>
-- Next step: find procedures for purifying different phage. Become experts on phage structure. possibly help with designing point mutations.
+- Discussed results from tittering experiment (preliminary experiment 1)
-:osmotic procedure
+: Contamination mostly likely resulted from the step involving suspending phage in LB – obvious contamination in the LB bottle
-:procedure from Dr. Grose
+: One of the stock phage solution had contamination as well
--Phage structure:
+- Discussed step of attack with Dr. Grose
-: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.
+: Decided to go with T7, if necessary Qbeta
-<br>
+: Need to learn to make top agar at various concentrations
-<font size="4"> '''3/25/13''' </font>
+: Need to do more background research and decide whether to assemble phage capsid in vitro (plasmid + E coli) or do direct mutagenesis of phage genome
--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.
+:: Need to correspond with the isolation team
--List of materials for Phage Purification Procedure from Dr. Grose
+- Sequencing will be for individual genes to cut down cost
-: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.
+: Need to design primers and get to know the genome of the phage
-:2. DNase I and RNase A from Bovine Pancreas (Roche or Cal- biochem). Stock solutions 1 mg/mL are stored at −20 ◦C.
+- Learnt about Mega5 to compare genome and protein sequence
-:3. Chloroform.
+<br>
-:4. Sodium chloride powder: NaCl ≥ 99. 5 % for molecular biology.
+<font size="4"> '''3/25/13''' </font>
-:5. Polyethylene glycol powder: PEG 6,000 (MW 5,000–7,000 g/mol) for molecular biology and biochemicalpurposes.
+- Reported on past week and plans for this week
+: From last week: titering experiment
+: This week
+:: Learn to make top agar at various concentrations
+:: Background research to determine in vitro assembly vs altering genome – look into specific techniques
+:: Comparing genome of phage and decide on possible site-directed mutagenesis options
-:6. Cesium chloride: CsCl ≥ 99. 9 % for density gradient purification.
+- Start working on designing our site directed mutagenesis
+: Qbeta vs MS2
+:: Look for places where sequences are significantly different
+:: Might be worthwhile to look at capsid structure to identify the regions where interactions between subunits take place
-:7. Ultracentrifuge equipments: Beckman L8-55M or equiva- lent.
+: Qbeta vs T7 major
+:: No real consensus – more worthwhile to compare capsid protein sequence of T7 with those that have similar size to it
-:8. Swinging-bucket rotors (Beckman): SW41 or SW28 and SW50 or SW65.
+: T7 major vs minor
+:: Minor is longer, but not necessary – the tail overhang is due to ribosome moving two codons downstream instead of three
+:: Suggest we can direct mutation to the poly-U site and prevent ribosome slippage
-:9. Centrifuge tubes (Beckman): thinwall or thickwall open-top polyallomer tubes:
+: Qbeta major vs minor
-::13.2 mL, 14 mm × 89 mm for the SW41 rotor.
+:: Just continue transcribing after it reaches the stop codon. What does the stop codon code for?
-::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.
+- Research into in-vitro assembly vs direct mutation of phage genome
+: It seems that we’ll need to clone the genome of the phage into a plasmid and let it assemble in an E coli
+: Using chemicals we can induce random mutations in phage – might be worthwhile if selection in agar is not working as well.
 <br>
@@ Line 179: / Line 134: @@
 <font size="4"> '''3/27/13''' </font>
-AC 3/27/2013
+- More research on genome of enterobacteria phage
-Found a great article on the structure and proteins of bacteriophage T4 http://www.farisaka.bio.titech.ac.jp/text/CMLS-Leiman-20031.pdf
+: Generation of the major and minor capsid in Q beta
-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:
+: Capsid protein information research
+: Capsid protein sequence comparison
-:GP-23
+- Outlined protocol for producing stock top agar
-::160 hexamers
+: [[Team:BYU_Provo/Notebook/SmallPhage/Winterexp/Period1/Exp/4.3 Top Agar Stock Preparation|4.3 Top Agar Stock Preparation]]
-: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)
 <br>
@@ Line 234: / Line 147: @@
 <font size="4"> '''3/29/13''' </font>
-DL 3/29
+- Worked on our first team presentation.
-	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
 <br>