Team:BYU Provo/Notebook/SmallPhage/Winterexp/Period3/Dailylog

From 2013.igem.org

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: [[Team:BYU_Provo/Small_Phage|Overview]]
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: <u> '''Small Phage''' </u> </font>
: [[Team:BYU Provo/Notebook/SmallPhage/Winterexp|March-April]]
: [[Team:BYU Provo/Notebook/SmallPhage/Winterexp|March-April]]
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- Made fresh LB and concentrated top agar stock
- Made fresh LB and concentrated top agar stock
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: [[Team:BYU_Provo/Notebook/SmallPhage/Winterexp/Period1/Exp/4.3 Top Agar Stock Preparation|4.3 Top Agar Stock Preparation]]
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: [[Team:BYU_Provo/Notebook/SmallPhage/Winterexp/Period3/Exp/4.3 Top Agar Stock Preparation|4.3 Top Agar Stock Preparation]]
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<br>
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<font size="4"> '''4/5/13''' </font>
<font size="4"> '''4/5/13''' </font>
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- Background research on phiX174: the only phage we have in stock
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- Performed spot test with E coli BL21 overnight liquid culture and WT T7 phage
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- Performed [[Team:BYU_Provo/Notebook/SmallPhage/Winterexp/Period1/Exp/3.20 Phage Viability Test|3.20 Phage Viability Test]]
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: [[Team:BYU_Provo/Notebook/SmallPhage/Winterexp/Period2/Exp/4.5 T7 phage viability assay|4.5 T7 phage viability assay]]
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- Created bacterial glycerol stock
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: 0.5mL of bacteria overnight liquid culture + 0.5mL of glycerol
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<br>
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<font size="4"> '''3/21/13''' </font>
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<font size="4"> '''4/6/13''' </font>
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- Checked up on results for [[Team:BYU_Provo/Notebook/SmallPhage/Winterexp/Period1/Exp/3.20 Phage Viability Test|3.20 Phage Viability Test]]
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- Checked up on [[Team:BYU_Provo/Notebook/SmallPhage/Winterexp/Period2/Exp/4.5 T7 phage viability assay|4.5 T7 phage viability assay]]
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<br>
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- 6:00pm overnight E coli liquid culture
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<font size="4"> '''3/22/13''' </font>
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: W3110 one colony in 25mL Erlenmeyer flask
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- Discussed results from tittering experiment (preliminary experiment 1)
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: BL21 one stab in 25mL Erlenmeyer flask
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: Contamination mostly likely resulted from the step involving suspending phage in LB – obvious contamination in the LB bottle
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- 9:00pm Streak bacteria culture to check for contamination and for storage
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: One of the stock phage solution had contamination as well
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: performed for both W3110 and BL21
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- Discussed step of attack with Dr. Grose
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<br>
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: Decided to go with T7, if necessary Qbeta
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<font size="4"> '''4/8/13''' </font>
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: Need to learn to make top agar at various concentrations
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- Performed [[Team:BYU_Provo/Notebook/SmallPhage/Winterexp/Period2/Exp/4.8 T7 phage viability assay 2|4.8 T7 phage viability assay 2]]
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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
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- Outlined final presentation content and divided up assignments
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:: Need to correspond with the isolation team
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<br>
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- Sequencing will be for individual genes to cut down cost
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<font size="4"> '''4/9/13''' </font>
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: Need to design primers and get to know the genome of the phage
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- Checked up on [[Team:BYU_Provo/Notebook/SmallPhage/Winterexp/Period2/Exp/4.8 T7 phage viability assay 2|4.8 T7 phage viability assay 2]]
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- Learnt about Mega5 to compare genome and protein sequence
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- Start overnight for E coli BL21
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<font size="4"> '''3/25/13''' </font>
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<font size="4"> '''4/10/13''' </font>
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- Reported on past week and plans for this week
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- Performed [[Team:BYU_Provo/Notebook/SmallPhage/Winterexp/Period2/Exp/4.10 T7 phage viability assay 3|4.10 T7 phage viability assay 3]]
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: From last week: titering experiment
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: This week
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:: Learn to make top agar at various concentrations
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:: Background research to determine in vitro assembly vs altering genome – look into specific techniques
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:: Comparing genome of phage and decide on possible site-directed mutagenesis options
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- Start working on designing our site directed mutagenesis
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- Looked into T7-family phage
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: Qbeta vs MS2
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:: Look for places where sequences are significantly different
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:: Might be worthwhile to look at capsid structure to identify the regions where interactions between subunits take place
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: Qbeta vs T7 major
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<br>
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:: No real consensus – more worthwhile to compare capsid protein sequence of T7 with those that have similar size to it
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: T7 major vs minor
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<font size="4"> '''4/11/13''' </font>
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:: Minor is longer, but not necessary – the tail overhang is due to ribosome moving two codons downstream instead of three
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:: Suggest we can direct mutation to the poly-U site and prevent ribosome slippage
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: Qbeta major vs minor
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- Check up on [[Team:BYU_Provo/Notebook/SmallPhage/Winterexp/Period2/Exp/4.10 T7 phage viability assay 3|4.10 T7 phage viability assay 3]]
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:: Just continue transcribing after it reaches the stop codon. What does the stop codon code for?
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- Research into in-vitro assembly vs direct mutation of phage genome
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- Worked on [[Team:BYU_Provo/Notebook/SmallPhage/Winterexp/Period3/PR| Presentation]]
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: It seems that we’ll need to clone the genome of the phage into a plasmid and let it assemble in an E coli
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: Using chemicals we can induce random mutations in phage – might be worthwhile if selection in agar is not working as well.
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<br>
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<font size="4"> '''3/27/13''' </font>
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<font size="4"> '''4/12/13''' </font>
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- More research on genome of enterobacteria phage
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: Generation of the major and minor capsid in Q beta
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- Cholera Presentation
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: Capsid protein information research
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: Capsid protein sequence comparison
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- Outlined protocol for producing stock top agar
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- Worked on [[Team:BYU_Provo/Notebook/SmallPhage/Winterexp/Period3/PR| Presentation]]
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: [[Team:BYU_Provo/Notebook/SmallPhage/Winterexp/Period1/Exp/4.3 Top Agar Stock Preparation|4.3 Top Agar Stock Preparation]]
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<br>
<br>
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<font size="4"> '''3/29/13''' </font>
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<font size="4"> '''4/13/13''' </font>
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- Worked on our first team presentation.
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- Continued working on [[Team:BYU_Provo/Notebook/SmallPhage/Winterexp/Period3/PR| Presentation]]
<br>
<br>

Latest revision as of 13:26, 9 September 2013


Small Phage March - April Notebook: April 1 - April 14 Daily Log



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

4/1/13

- Presentation day!

Presentation


4/3/13

- Learned about how to start an overnight liquid culture for host bacteria

- Learned about how to create bacterial glycerol stock

- Made fresh LB and concentrated top agar stock

4.3 Top Agar Stock Preparation


4/4/13

- T7 arrived

- Overnight liquid culture for bacterial host started by Dr. Grose


4/5/13

- Performed spot test with E coli BL21 overnight liquid culture and WT T7 phage

4.5 T7 phage viability assay

- Created bacterial glycerol stock

0.5mL of bacteria overnight liquid culture + 0.5mL of glycerol


4/6/13

- Checked up on 4.5 T7 phage viability assay

- 6:00pm overnight E coli liquid culture

W3110 one colony in 25mL Erlenmeyer flask
BL21 one stab in 25mL Erlenmeyer flask

- 9:00pm Streak bacteria culture to check for contamination and for storage

performed for both W3110 and BL21


4/8/13

- Performed 4.8 T7 phage viability assay 2

- Outlined final presentation content and divided up assignments


4/9/13

- Checked up on 4.8 T7 phage viability assay 2

- Start overnight for E coli BL21


4/10/13

- Performed 4.10 T7 phage viability assay 3

- Looked into T7-family phage


4/11/13

- Check up on 4.10 T7 phage viability assay 3

- Worked on Presentation


4/12/13

- Cholera Presentation

- Worked on Presentation


4/13/13

- Continued working on Presentation


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