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

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| colspan="3" | <font color="#333399" size="5" font face="Calibri"> '''Large Phage March - April Notebook: March 15 - March 31 Daily Log'''</font>
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| colspan="3" | <font color="#333399" size="5" font face="Calibri">  
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: '''Small Phage March - April Notebook: March 15 - March 31 Daily Log'''</font>
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<font color="#333399" size="3" font face="Calibri">
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: [[Team:BYU_Provo/Small_Phage|Overview]]
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<font size = "4">
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: [[Team:BYU Provo/Notebook/LargePhage/Winterexp|March-April]]
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: <u> '''Small Phage''' </u> </font>
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: [[Team:BYU Provo/Notebook/LargePhage/Springexp|May-June]]
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: [[Team:BYU Provo/Notebook/SmallPhage/Winterexp|March-April]]
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: [[Team:BYU Provo/Notebook/LargePhage/Summerexp|July-August]]
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: [[Team:BYU Provo/Notebook/SmallPhage/Springexp|May-June]]
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: [[Team:BYU Provo/Notebook/LargePhage/Fallexp|September-October]]
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: [[Team:BYU Provo/Notebook/SmallPhage/Summerexp|July-August]]
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: [[Team:BYU Provo/Notebook/SmallPhage/Fallexp|September-October]]
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<font size="4"> '''3/15/13''' </font>
<font size="4"> '''3/15/13''' </font>
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3/15/13 - Friday - Bryan Merrill (BDM)
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-Today we began research on phage purification
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Spreadsheet
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for plasmids, primers, and bacterial strains (and phage)
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Notes
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on what we found:
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Largest:
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Phage G, lytic, capsid, BSL1, generation time = 40 mins.
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Smallest:
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RH1 infects Rhodococcus, capsid size of 43 nm, 50kb?
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Smallest
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for E. coli: Spreadsheet (Jade) MS2, capsid protein size 137 aa, 27 nm.
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Well
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charaterized, Qbeta as well
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Dissertation
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-Focused on T4 and T7 as this is what the other groups will be focusing on
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on capsid design – do longer protein sequences correspond to bigger
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:-T7 can self assemble with the help of scaffolding proteins without forming procapsids
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capsids?  Are bigger capsids built from
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:-T4 assembles using procapsids
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bigger monomers or more monomers?
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-Some phage can have their DNA polymerase genes knocked out to make a hollow phage
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T7 – list of all genes, Gp10a and Gp10b (major and minor capsid proteins)
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:-Possibility for making ghost capsids
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Read the thesis/dissertation –
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<br>
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3 Groups:
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Large phage
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Small phage
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How to build the capsids (heat shock or self-assembly)
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-
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Find phage which are well-characterized
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-
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Find:
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How
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phage are used for drug delivery (applications)
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Find out how capsid sizes change and mutate (targeted mutagenesis)
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Start by selecting, move towards targeted mutagenesis
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What are the applications for phage already
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Cloning, sequence, and expression of the temperature-dependent phage T4 capsid assembly gene 31
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Modulation of bacteriophage T4 capsid size.
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-
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 +
<font size="4"> '''3/16/13''' </font>
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Goal:
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- Created a priority list of things we need to complete:
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Build a library of multiple sizes of phage capsids and
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:-Find out how drugs are put into capsids
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provide tools to assemble these phage capsids so they can be used for drug
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:-Possibly contact F. W. Studier for his amber T7 phage strain
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delivery and nanotechnology
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::-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>
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Look
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<font size="4"> '''3/18/13''' </font>
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into:
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Metabolic
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load on bacteria based on capsid size?
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Screening
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- Presented current understanding and plans for the future
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methods:
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: Decided on a two-focus attack
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Increasingly
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:: Evolution – selecting naturally occurring smaller phages
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harsh environments, less nutrients, smaller phage?
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:: Site directed mutagenesis – using genome info and comparative studies to identify sites to target
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Using
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- More background research
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ozone for selection
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: Possible ways of making phage smaller
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3/15/13-Keltzie Smith
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<br>
-
Today we narrowed in on the phage we are going to use as a starting point for expansion. We found that it wasn’t as easy as we thought to simply look up what would be best for us since there are bits information here and there, but not all in the same location- I suppose this is why we are doing the research that we are- filling a gap! In deciding which to use we took into account genome size as well as how characterized it is. We decided that T4 is the simplest and most effective route to take even though it is technically not the largest phage that infects E. coli.
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 +
<font size="4"> '''3/20/13''' </font>
-
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- Background research on phiX174: the only phage we have in stock
 +
- Performed [[Team:BYU_Provo/Notebook/SmallPhage/Winterexp/Period1/Exp/3.20 Phage Viability Test|3.20 Phage Viability Test]]
 +
<br>
-
+
<font size="4"> '''3/21/13''' </font>
 +
- Checked up on results for [[Team:BYU_Provo/Notebook/SmallPhage/Winterexp/Period1/Exp/3.20 Phage Viability Test|3.20 Phage Viability Test]]
 +
<br>
 +
<font size="4"> '''3/22/13''' </font>
 +
- 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
-
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- 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
-
Large phage procedures:
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: 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
-
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: Need to design primers and get to know the genome of the phage
-
 
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- Learnt about Mega5 to compare genome and protein sequence
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Paper about UV light and backup genes?
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Decreasing agar concentration – select for
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small plaque sizes
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Find T4 phage and host
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Start growing them (archive sample of the
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phage)
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Determine sequence of the relevant capsid
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proteins
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Mutate them
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Determine the sequence again
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Find a way to create just capsids of the larger
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size
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<br>
<br>
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<font size="4"> '''3/18/13''' </font>
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<font size="4"> '''3/25/13''' </font>
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3/18/13-Keltzie Smith
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-
As of right now our main priority is making sure the T4 we have now is working. Today we started our K12  E.Coli. Next time we’ll see if our T4 is viable, once we see plaques on our plates our next step will be to sequence our phage to determine what we have to start out with an we’ll mutate from there. One thing we are hoping to look deeper into is UV irradiation to select for bigger bacteriophages.
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-
18 March 2013 - BDM
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- Reported on past week and plans for this week
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18 March 2013 – Monday – BDM
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: From last week: titering experiment
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Notes:
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: This week
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Cholera group
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:: Learn to make top agar at various concentrations
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Biofilm – grow a cholera biofilm in the lab to test constructs that have already been created
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:: Background research to determine in vitro assembly vs altering genome – look into specific techniques
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Plasmid prep – sequencing inserts in plasmid to see if the genes were inserted correctly.
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:: Comparing genome of phage and decide on possible site-directed mutagenesis options
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2 membrane proteins, 2 phosphorylation proteins, 2 genes turned on (RFP and GFP), small RNAs. Need to clone in protein interaction and siRNA degradation.
+
-
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Phage group
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Protein prep/capsid purification – T7 proteins, requires scaffold protein and capsid proteins to assemble. Other phage, sometimes this doesn’t happen. T4 forms procapsids, but not large ones in vitro.  No DNA in capsids – knock out polymerases. Capsid size can depend on environmental factors.
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Small phage – Directed and random mutagenesis
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-
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Genetic Control of Capsid Length in Bacteriophage T4
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Genetic studies on capsid-length determination in bacteriophage T4
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Evolution of Phage Capsid and Genome Size
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The Capsid of the T4 Phage Superfamily
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-
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Hendrix, R.W. (2009) Jumbo bacteriophages. Curr. Top. Microbiol. Immunol. 328:229-240
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-
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Lee, K.K., L. Gan, H. Tsuruta, C. Moyer, J.F. Conway, R.L. Duda, R.W. Hendrix, A.C. Steven, and J.E. Johnson (2008) Virus capsid expansion driven by the capture of mobile surface loops. Structure 16:1491-1502
+
 +
- 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
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<br>
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: Qbeta vs T7 major
 +
:: No real consensus – more worthwhile to compare capsid protein sequence of T7 with those that have similar size to it
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<font size="4"> '''3/20/13''' </font>
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: 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
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3/20/13-Keltzie Smith
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: Qbeta major vs minor
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Today we learned about phage titering.
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:: Just continue transcribing after it reaches the stop codon. What does the stop codon code for?
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pfu's are plaque forming units. We tried to see how competent our phage was, unfortunately we ran into quite a few problems. Much of it due to our lack of experience. We put 10 microliters of phage with 90 microliters of LB and did a 1:10 dilution series. we put 20 microliters of each of the dilution series with .5mLs of E.coli. After we let a twenty minute incubation period pass we added 5 mLs of top agar and plated the phage. With the way everything went, I would imagine there will be a lot of contamination, but I certainly learned quite a bit about the process. Hopefully we see some good growth!
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-
�
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20 March 2013 - BDM
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Procedure Description - Titering all stock samples of E. coli phage from Dr. Breakwell’s fridge
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Purpose - See which phage stocks are viable, and how many PFUs/mL they have.
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Theoretical
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Protocol - We performed a 1:10 dilution series (10^0 to 10^-5) on each of the stock samples. We infected 0.5 mL of an overnight growth of E. coli K12 with 20 uL of each dilution series tube. After infecting for 20 minutes, we added 5 mL of 1x LB top agar and plated them on LB plates. We let them incubate at 37C overnight.
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I also tried a spot test of each sample on an E. coli lawn (to check for plaque formation) and on a plain LB plate (to check for phage lysate contamination).
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Results
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Actual
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Protocol - We performed this experiment as we planned on doing it. The agar frequently boiled over in the microwave which made it difficult to use.Next time we will use 2X and mix it right before we need it.
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Results - None of the samples showed signs of plaques after 24 hours. I will let them incubate one more night. Three of the phage lysates were contaminated.
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Observations - It is very likely that these phage samples are no longer viable. We ought to make a list of which phages we would like from Dr. Casjens at U of U and start from fresh samples instead of these ones.
+
-
 
+
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<br>
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-
 
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-
<font size="4"> '''3/22/13''' </font>
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-
 
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22 March 2013 - Friday - BDM
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Procedure description - Check plates for any plaques that appeared during the second 24 hours.
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Identify phage samples we need to request from Dr. Casjens at U of U and send the list to Dr. Grose.
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We still saw no plaques and a lot of contamination. We’re going to get Dr. Grose a list of the phage we want... probably just T4.
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-
 
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3/22/13 Keltzie Smith
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Today we observed the phage that we plated on Wednesday. There was very little growth of anything so we decided to order some from a professor who has the ones we need. Also Bryan gave us a great tutorial on how to use different programs to compare protein and dna sequences. From here we are going to try to get to  know the structure of the phage to better understand how to make point directed mutations.
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<br>
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<font size="4"> '''3/25/13''' </font>
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3/25/13 Keltzie Smith
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http://www.virologyj.com/content/7/1/356
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Today we continued research on the t4 capsid. From the article above we discovered that t4 has at least three essential capsid proteins. gp23, gp24 and gp20. With the folding of these essential proteins, chaperone protein systems are involved. For the folding of gp 23 GroEL chaperonin system and phage co-chaperonin gr31 are required. On the capsid of t4 Hoc and Soc “decorate the outside. Hoc(highly antigenic outer capside protein) is a dumbbell shaped monomer at the center of each gp23 hexon, Soc (small outer capsid protein) is a rod shaped moleule that binds between gp 23 hexons. Both Hoc and Soc bind to the capsid after its completion.
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-
 
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25 March 2013 - BDM
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25 March 2013 – Monday
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Large Phage
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Rational design (mutagenesis)
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        Dependent on what we know
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Genetic selection (UV, agar, etc.)
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        Ask a question, results tell the answer
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Small phage
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Vary top agar concentrations to select for smaller phage.
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Plate T7 on various concentrations of agar; where it fails to form a plaque, start there
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Purification
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Found papers for phage purification
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Liquid culture as well
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Nanotechnology applications
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Today: list of reagents
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Cholera phage and locations:
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Outbreaks in poorly treated water; Africa, S. America, etc.
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Most US cases came from travel
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Find cholera phage
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Biofilms
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Proteosomes – degrade proteins
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Amylases – degrade polysaccharides
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All biofilms are different
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Sample colonies and determine ratios in the biofilm
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Plant extracts worked
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Start growing cholera, see if biofilm comes (incubate at various temperatures)
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-
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Recipes:
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LB 2x top agar
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10g LB broth
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4g agar
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-
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10 of 16 genes for prohead formation are essential.
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GroEL (host-encoded)
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T4 gp31 can substitute for GroES which binds to GroEL
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-
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GroEL-gp31 helps gp23 fold (major capsid protein)
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-
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gp22 and gp21 is part of the scaffold, as well as gp23 - assembled onto initiator (portal vertex) which is 12-mer of gp20 in a ring.
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-
+
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Pentamers of gp24 are placed at other 11 vertices.
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-
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T4 protease (gp21) degrades scaffold and cleaves head proteins (23 and 24) creating space.)
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-
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DNA packaging forms the initiated small particle
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 +
- 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>
<br>
Line 276: Line 139:
<font size="4"> '''3/27/13''' </font>
<font size="4"> '''3/27/13''' </font>
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3/27/13 KS
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- More research on genome of enterobacteria phage
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I am going to be going deeper into studying about t4 capsid assembly including all the proteins that are involved in it. Hopefully this will give a better look into how we can possibly change things to make a bigger capsid.
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-
27 March 2013 - BDM
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: Generation of the major and minor capsid in Q beta
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27 March 2013 – Wednesday
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: Capsid protein information research
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Gp40 embeds in membrane
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: Capsid protein sequence comparison
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Gp20 attaches to Gp40
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Tons of gpalt, gp21, gp67, gp68, IPI, IPII, IPIII attach
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-
Gp22 surrounds it
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Gp23 and 24 coat the inside
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Gp21 is activated, everything breaks down and disperses
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-
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Giant capsids are produced under heavy mutagenic pressure, UV irradiation, and always have multiple genomes in them.  Mutagens typically have mutations in gp23.
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-
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To do:
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Compile list of reagents needed to mutagenize large phage
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Put together presentation about the process of doing this
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 +
- 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>
<br>
Line 299: Line 152:
<font size="4"> '''3/29/13''' </font>
<font size="4"> '''3/29/13''' </font>
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3/29/13 KS
+
- Worked on our first team presentation.
-
Today we spent class researching for our presentations for Monday. I have been working on the mechanism that t4 uses to package the DNA to see how we can take extra DNA and have the t4 encapsulate it to create a larger phage that we want. This is also an important thing to know concerning how they package medicines in there.
+
-
 
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Friday 29 March 2013- BDM
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I prepared my part of the presentation for Monday before class today.  I missed most of class but was excused by Dr. Grose.
+
<br>
<br>

Latest revision as of 13:23, 9 September 2013


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



Small Phage
March-April
May-June
July-August
September-October

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.


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