Team:BYU Provo/Notebook/SmallPhage/Fallexp/10.18 Cloning T7 Capsid Protein into iGEM Registry

Small Phage

March-April

May-June

July-August

September-October

Clone both WT and mutant T7 Capsid Protein into iGEM registry

II) Expected Outcome

• WT and mutant T7 capsid protein cloned into the iGEM registry.

III) Reagents Used

• Reagents required for cloning: dNTP, Phusion polymerase, butter, ddH2O, etc.

• Mutant phage S4, S10, S21, and L8; WT phage

IV) Procedure

1) Amplification and purification of insert (10.18)

I. DNA purification

* Put 5 ul of phage sample (S4, S10, S21, L8, and WT) and 45 ul of ddH20 into in PCR tubes with respective labeling.

* Boil for 12 minutes in the PCR machine (98 Celsius).

* Remove the tubes from the PCR machine and keep on ice until use in PCR.

2) 2) Propagating Mutant Phage (10.10)

• Three different conditions of propagation was used for WT, S4, S10, and L8

- Erlenmeyer flask: 10 mL LB + 1 mL E coli B liquid culture overnight + 10 uL of phage

- 15 mL centrifuge tube: 5 mL LB + 0.5 mL E coli B liquid culture overnight + 10 uL of phage

- autoclaved test tube: 1 mL of LB + 100 uL E coli B liquid culture overnight + 10 uL of phage

• Wild-type and mutant phages were allowed to propagate for approximately 24 hours before purified via centrifugation and choloroform.

• Spot tests at -2, -4, -6, and -8 was performed for each sample to estimated phage concentration after propagation.

• Each phage sample was then plated at -7 using x4 agar to verify the mutants' phenotypic stability after propagation.

3) TEM (10.16)

• Phage Purification Team set up copper grids and arranged for TEM appointments to look at S4, S10 and L8

4) Sequencing (10.17-10.?)

• During the week when we were waiting for the new sequencing primers (BI319 and BI320) to arrive, we where able to isolate a new mutant phage S21. Thus, we will sequence S4, S10, S21, L8, and WT T7 phage to map out mutations altering phage capsid sizes.

• DNA isolation, PCR, and gel electrophoresis protocol was similar to that of 5.20 T7 Minor Capsid Protein PCR

Note for sequencing we used the primers BI257 and BI258.

V) Results

2) Propagating Mutant Phage

• Plaque formed up to -6 for each sample, implying a concentration of at least 10E8 PFU/mL

• Propagated phage retained their larger or smaller capsid size as confirmed by their smaller and larger plaque sizes at x4 agar.

3) TEM ADD PICTURE OF PLATE

VI) Conclusion