Team:Rice University/Notebook/20 June 2008

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**Lysis Assay of VCS257 SupA+  
**Lysis Assay of VCS257 SupA+  
*#Cells were grown O/N @ 37*C in Lambda media.   
*#Cells were grown O/N @ 37*C in Lambda media.   
-
*#4.5mL culture was infected with 4.5uL of phage packaging product from November, 2007 and allowed to incubate for 20min @ 37*C shaking @ 250 rpm.  Infected culture was heat shocked @ 42*C for 20min, inducing lysis by denaturing the CI857 repressor.   
+
*#4.5mL culture was infected with 4.5uL of phage packaging product from November, 2007 and allowed to incubate for 20min @ 37*C shaking @ 250 rpm.   
 +
*#Infected culture was heat shocked @ 42*C for 20min, inducing lysis by denaturing the CI857 repressor.   
*#Cells were then grown and their optical density was measured at different time points after heat shock.
*#Cells were then grown and their optical density was measured at different time points after heat shock.
-
**Lysogeny Screen - Results
+
*#'''Result'''-lysis was never observed. Cells may need to be maintained at higher temperature to ensure all phage infections result in lysis.
-
*#An infected VCS257 culture was streaked onto LB-agar.  Isolated colonies were used to innoculate a 96 deep-well plate which was grown O/N @ 30*C shaking @ 300rpm.  Using the well replicator, each of the wells was spotted onto two large petri dishes.  One dish was grown @ 30*C and the other was grown @ 42*C O/N.  Spots that grew on 30*C plates and not on 42*C plates are lysogens as the phage infected cells would lyse due to the temperature sensitive cI.  The plates show that there is only one colony that could be a possible lysogen.  
+
**Lysogeny Screen
 +
*# '''Result'''-An infected VCS257 culture was streaked onto LB-agar.  Isolated colonies were used to innoculate a 96 deep-well plate which was grown O/N @ 30*C shaking @ 300rpm.  Using the well replicator, each of the wells was spotted onto two large petri dishes.  One dish was grown @ 30*C and the other was grown @ 42*C O/N.  Spots that grew on 30*C plates and not on 42*C plates are lysogens as the phage infected cells would lyse due to the temperature sensitive cI.  The plates show that there is only one colony that could be a possible lysogen.  
*# The possible lysogen colony was streaked onto LB plates to be grown O/N @ 30*C and 42*C to verify temperature screen.  
*# The possible lysogen colony was streaked onto LB plates to be grown O/N @ 30*C and 42*C to verify temperature screen.  
*# A colony PCR was performed on the possible lysogen using the CroF and CroR primers.  If a copy of the lambda genome is incorporated into the lysogen's, the PCR should result in amplification of the phage gene ''cro'' and produce a 400bp product.  Additionally, three other PCR reactions were performed in parallel.
*# A colony PCR was performed on the possible lysogen using the CroF and CroR primers.  If a copy of the lambda genome is incorporated into the lysogen's, the PCR should result in amplification of the phage gene ''cro'' and produce a 400bp product.  Additionally, three other PCR reactions were performed in parallel.
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*## -positive control using lambda DNA
*## -positive control using lambda DNA
*## -second sample using lambda particles from stocks
*## -second sample using lambda particles from stocks
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*#'''Result'''-PCR gel analysis shows that the bacterial colony, phage DNA, and lambda particles all produce a PCR product at approximately 440bp, where the amplified ''cro'' should be.  This data further supports the conclusion that the screened colony is a lysogen, and that it is possible to use packaged phage DNA as a template in a PCR.
 +
*#'''Result'''-Temperature screen of the colony showed that streaked plates did not grow colonies @ 42*C and grew an abundant number @ 30*C.
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[[Image:lysogeny screen.jpg|400px]]
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{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"
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!align="center"|[[Team:Rice_University|Home]]
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!align="center"|[[Team:Rice_University/Team|The Team]]
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!align="center"|[[Team:Rice_University/Project|The Project]]
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!align="center"|[[Team:Rice_University/Parts|Parts Submitted to the Registry]]
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!align="center"|[[Team:Rice_University/Modeling|Modeling]]
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Latest revision as of 20:27, 25 June 2008

Friday, 20 June

  • Taylor Stevenson:
    • Lysis Assay of VCS257 SupA+
    1. Cells were grown O/N @ 37*C in Lambda media.
    2. 4.5mL culture was infected with 4.5uL of phage packaging product from November, 2007 and allowed to incubate for 20min @ 37*C shaking @ 250 rpm.
    3. Infected culture was heat shocked @ 42*C for 20min, inducing lysis by denaturing the CI857 repressor.
    4. Cells were then grown and their optical density was measured at different time points after heat shock.
    5. Result-lysis was never observed. Cells may need to be maintained at higher temperature to ensure all phage infections result in lysis.
    • Lysogeny Screen
    1. Result-An infected VCS257 culture was streaked onto LB-agar. Isolated colonies were used to innoculate a 96 deep-well plate which was grown O/N @ 30*C shaking @ 300rpm. Using the well replicator, each of the wells was spotted onto two large petri dishes. One dish was grown @ 30*C and the other was grown @ 42*C O/N. Spots that grew on 30*C plates and not on 42*C plates are lysogens as the phage infected cells would lyse due to the temperature sensitive cI. The plates show that there is only one colony that could be a possible lysogen.
    2. The possible lysogen colony was streaked onto LB plates to be grown O/N @ 30*C and 42*C to verify temperature screen.
    3. A colony PCR was performed on the possible lysogen using the CroF and CroR primers. If a copy of the lambda genome is incorporated into the lysogen's, the PCR should result in amplification of the phage gene cro and produce a 400bp product. Additionally, three other PCR reactions were performed in parallel.
      1. -negative control using water
      2. -positive control using lambda DNA
      3. -second sample using lambda particles from stocks
    4. Result-PCR gel analysis shows that the bacterial colony, phage DNA, and lambda particles all produce a PCR product at approximately 440bp, where the amplified cro should be. This data further supports the conclusion that the screened colony is a lysogen, and that it is possible to use packaged phage DNA as a template in a PCR.
    5. Result-Temperature screen of the colony showed that streaked plates did not grow colonies @ 42*C and grew an abundant number @ 30*C.

Lysogeny screen.jpg



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