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

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Latest revision as of 13:23, 9 September 2013

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

Small Phage

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 16: @@
 <font color="#333399" size="3" font face="Calibri">
-: [[Team:BYU_Provo/Phage_Purification|Overview]]
+<font size = "4">
-: [[Team:BYU Provo/Notebook/Phage_Purification/Winterexp|March-April]]
+: <u> '''Small Phage''' </u> </font>
-: [[Team:BYU Provo/Notebook/Phage_Purification/Springexp|May-June]]
+: [[Team:BYU Provo/Notebook/SmallPhage/Winterexp|March-April]]
-: [[Team:BYU Provo/Notebook/Phage_Purification/Summerexp|July-August]]
+: [[Team:BYU Provo/Notebook/SmallPhage/Springexp|May-June]]
-: [[Team:BYU Provo/Notebook/Phage_Purification/Fallexp|September-October]]
+: [[Team:BYU Provo/Notebook/SmallPhage/Summerexp|July-August]]
+: [[Team:BYU Provo/Notebook/SmallPhage/Fallexp|September-October]]
 </font>
@@ Line 32: / Line 36: @@
 <font size="4"> '''3/15/13''' </font>
-- TODAY MARKS THE START OF THE PHAGE PURIFICATION TEAM!
+-Today we began research on phage purification
-- 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.
+-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>
-- Important findings
+<font size="4"> '''3/16/13''' </font>
-:Phage have been purified before
-:Phage capsids can self assemble
+- Created a priority list of things we need to complete:
-:Phage (amber strain) can have their genes knocked out to make a hollow phage
+:-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
 <br>
@@ Line 45: / Line 58: @@
 <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 69: @@
 <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 83: @@
 <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.
+- 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.
-:11. Dialysis tubing: Spectra/Por molecular-porous membranetubing, MWCO 12–14,000.
+<br>
-: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.
+<font size="4"> '''3/27/13''' </font>
-:9. Supernatant from number 8.
+- More research on genome of enterobacteria phage
-:10. Residue from number 8 dissolved in saline
+: 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
+: [[Team:BYU_Provo/Notebook/SmallPhage/Winterexp/Period3/Exp/4.3 Top Agar Stock Preparation|4.3 Top Agar Stock Preparation]]
 <br>