Team:BYU Provo/Notebook/SmallPhage/Fallexp/10.8 Characterization of Mutant Phage


Small Phage September - October Notebook: Experiments

Small Phage

10.8 Characterization of Mutant Phage

I) Purpose

To characterize the mutant phage we selected from 9.13 Mutagen Concentration Test

II) Expected Outcome

  • EM pictures of phage with distinctively larger or smaller size.
  • Sequencing results that map out mutations that induce T7 phage capsid size changes.

III) Reagents Used

  • Mutant phage S4, S10, S21, and L8
  • x8 top agar
  • LB

IV) Procedure

1) Overview of previous attempts before iGEM Regional Jamboree

  • Before the Regional Jamboree, we identified our S4, S10, and L8 mutant. We tried to sequence their capsid genes (with the help of Dr. Grose) and take pictures using TEM. Unfortunately, the sequencing results' accuracy was less than 20%. And our phage does not have a high enough titer to be seen under electron microscope.
  • Thus we started off our characterization procedures with designing new primers (BI319 and BI320) and propagating our mutant phage.

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.

VI) Conclusion

  • We have taken several EM's of the phage, but have yet to get results for their capsid sizes. We hope to accomplish this before we leave for the jamboree.