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- Small Phage
- March-April
- May-June
- July-August
- September-October
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10.18 Cloning T7 Capsid Protein into iGEM Registry
I) Purpose
- 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.
- 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
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