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

Overview

March-April

May-June

July-August

September-October

• all work was done in yellow light?

chloroform calcium chloride magnesium chloride

To do: grow up T1 and T4

4/5/13

4/5/13-KS and BDM Today we did a spot test with 11T4, T4Do(s), 12T4r, 40T4r, 40T4 with e.coli BL21. Procedure: We plated .5 mL of an overnight culture of E. coli BL21 in 4.5 mL of LB 1x top agar. When it had hardened, we spot tested 5 uL of the following samples on the plates: 11T4, T4Do(s), 12T4r, 40T4r, 40T4 into 6 sectors of a plate. T4Do came from Dr. Casjens at the University of Utah. We incubated these plates at 37 degrees Celsius. We plated a control of just bacteria. Also, we infected .5 mL of BL21 with 1 uL of the T4Do sample for 20 mins, and then plated it. We started two liquid cultures, each containing 1 mL of BL21. One received 1 uL of T4Do, the other received 10 uL. These were incubated on a shaker at 37C.

4/8/13

We performed a phage titer spot test on two bacteria. We used the BL21 and W3110 strains of E. coli. We first put 100 microL of broth into epindorf tubes. We then took 10 microL of 1L, 10L, 40T4, T4DOS, and T4 infected phage and placed it in separate tubes labeled -1. We performed a dilution series taking out 10 microL each time. The last tube we did not remove 10 microL so the total volume was 110 microL. Next we added 4.5 mL of 1x top agar to .5mL of bacteria for both strains and plated it on LB plates. We had previously divided the plates into six sections with the labels 0, -1, -2, -3, -4, and -5. We took the phage from each concentration and spotted it on the plates. We allowed the plates to sit and let the phage soak in. We then placed the plates in the 37 C overnight.

4/10/13 Results (Wed. 4/10/13) We saw phage plaques in all spots (10^0 through 10^-5) on all bacteria for 10L (10uL of T4Do stock in 1 mL of bacteria grown over the weekend), 1L (1uL same as above), T4Do (stock), and T4 infected plate flood. 40T4 from the fridge grew on BL21 but not W3110. I had started two liquid cultures of 100uL of liquid from the plate flood in 1mL of W3110 and BL21. The W3110 bacteria was visibly lysed (clear); the BL21 bacteria had some snot but was not lysed.

Procedure We pelleted the liquid from the two liquid cultures and did a 10^0 through 10^-5 dilution on each, along with a bacterial lawn control. We also started two more liquid cultures of 1 mL from the plate flood in 10 mL of W3110. We will shake these in a 100 mL flask at 37C.

4/12/13

- 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.