Template:Team 5 Notebook

Day 1

--Jennifer Lee 16:30, 12 April 2010 (EDT)
ZFN device not ready.

Transform cells with ZFN-selflysis devices. Also transform cells with 9145-sbb23 plasmid to grow up excess plasmid.
Transform 0.5uL plasmid + 10uL cells. Add 2.5uL water. Add 1.5uL KCM. Heat shock for 90 sec, allow 10 min recovery.

Prep atc dilution.

Day 2

--Jennifer Lee 18:19, 14 April 2010 (EDT)
DNA Stability Test assay: Template:DNA_Stability_Test_Protocol

pBca9145 digestion. (ask Jeni)

• Digest 12uL plasmid DNA with 0.5uL AlwN1 enzyme and 1uL NEB buffer. Incubate at 37C for an hour.--Done at 3pm
  
• Zymo gel purify digested plasmid. Template:SBB-Protocols_Zymo3 Store for next Monday 4/19/2010.
  
• ResultClean single band ~2500-3000bp cut out of gel and stored in 600uL ADB buffer.
  

Mini prep the sbb23 plasmid from cultures (ask Ben). Store DNA for ZFN enrichment assay.

Atc dilution

• Raffi prepped a 1,000X atc dilution.
  

Day 3

--Jennifer Lee 19:18, 19 April 2010 (EDT) Continue stability test assay: Template:DNA_Stability_Test_Protocol

• Prep cells for lysing (spin down and wash).
  
• Remake atc dilution: Team 2 diluted in water and found that a 100,000X dilution was not enough to induce lysis and that a 1,000X dilution would be better. We will remake the atc solution, diluting into NEB buffer so we don't need to add more NEB to the cells.
• --450uL ddH2O, 50uL 10X NEB and 0.5uL 1X atc (2mg/mL).
• Perform stability test, up to inactivation point, with AlwN1 digested pBca9145 plasmid.
  
• Run gels to determine product size.
  

1. ladder
2. negative control (lysate only, no plasmid)
3. Timepoint 0
4. TP 7
5. TP 10
6. TP 15
7. TP 20
8. TP 30
9. TP 45
10. TP 60
11. ladder

Result Based on the gel, we determined that this experiment failed. By discussing the lysis step with Raffi's group, we determined that the cells are not lysing after all and will try to use an alternate plasmid.

Day 4

--Jennifer Lee 19:18, 21 April 2010 (EDT)

Because the self-lysis device did not work, we are being given a new self-lysis plasmid.

• Mini-prep BamHI enzyme containing plasmid (ampR) from cells. Transform with the BamHI plasmid and new self-lysis plasmid (that has specR and BamHI site) at a 1:1 ratio (1uL). Template:SBB-Protocols_Micro1, but divide everything by 20.
  
• Plate on spec/amp plate
  

• New plan:
  
  • Want to redo lysis with 1mL total volume in a test tube at 37C, shaking. Induce lysis with 100X atc. Do this in both LB and NEB. Re-run gel
    

Day 5

--Jennifer Lee 19:00, 26 April 2010 (EDT) Induce self-lysis of cells containing only self-lysis plasmid. 1mL total volume in a test tube at 37C, shaking. Induce lysis with 100X atc. Do this in both LB and NEB.

• Spin down self-lysis cells, resuspend in LB and NEB according to the next bullet:
• 1mL of LB or NEB plus 10uL 1X atc (2mg/mL) which gives us atc at a concentration of 20ug/mL
  
• Set in 37C incubator, 1 hour.--Done at 3:15pm
  
• Result Cannot determine successful self-lysis based on color. We should have run negative controls without atc for both NEB and LB. Decided to run gel of the supernatant to perhaps see whether genomic DNA will appear in the gel.
• Run gel of lysate supernatant (spin down):
  
• 
  1. ladder
  2. LB
  3. NEB
     

Gel Results The gel appears to display some sort of buffer problem, indicated by the blue smile. Because we did not run a control for this experiment (cells with no atc induction) we cannot say whether self-lysis occurred. Therefore, we cannot say much of this version of the protocol, and instead we will take Team 2's successful induction of lysis and replicate their protocol on Wednesday, 4/28/2010.

Digest sbb23 plasmid (only need the plasmid to contain AlwN1 enzyme and BamHI sites).

• Digest 12uL plasmid DNA with 0.5uL AlwN1 enzyme and 1uL NEB buffer. Incubate at 37C for an hour.--Done at 3:10pm
  
• Zymo gel purify digested plasmid. Template:SBB-Protocols_Zymo3
  
• 
  1. Ladder
  2. sbb23 plasmid AlwNI digestion
• Cut out top band, ~3000bp
  

Gel Results With successful digestion, we expect a band of approximately 3000bp, which we saw with the top band in this gel. However, we did not expect the bottom band, which may be product of an incomplete or failed digestion. Because of this discrepancy in expected and obtained results, we will likely perform a digestion of an alternate plasmid Wednesday, while inducing self-lysis.

Day 6

To do: perform self-lysis using Raffi's protocol, run a concurrent digestion of pBjk2741-Bca1144 plasmid with EcoRI; perform stability test on the linearized pBjk2741-Bca1144 plasmid.

BamHI transformation of cells failed, so we cannot perform the actual assay. Can only run the stability test.

Lysis

• Successful lysis confirmed Day 5 in Raffi's group (they ran a gel to determine whether cells lysed under atc induction). We will perform the same experiment, see protocol at Team_2_Notes#4.2F26.2F10_Wetlab_Notes:_Self-Lysis_test.
  
• Add 10uL 100X atc directly to cell culture tube (LB media) and lyse in shaking incubator at 37C for an hour. --Done at 2:45pm
  
• Run gel with 8uL of lysate supernatant and 1uL 10X loading buffer. Land with atc should have significantly more DNA than the lane without (control). If we can distinguish the DNA amounts, we can confirm that self-lysis occurred.
  
• 
  1. Ladder
  2. No atc (control)
  3. Atc
• Gel result From the gel, it is clear that we see genomic DNA in the atc lane but no genomic DNA in the 'no atc' lane. This result indicates that atc-induced self-lysis of the cells was successful.

Plasmid digestion

• Digest 12uL plasmid DNA with 0.5uL EcoRI enzyme and 1uL NEB buffer. Incubate at 37C for an hour.--Done at 2:30pm
  
• Zymo gel purify the plasmid via Template:SBB-Protocols_Zymo3. Load all on the gel with 1uL 10X loading buffer. Cut out 3080bp sized band.
  
• 
  

1. Ladder
2. EcoRI digest of pBjk2741-Bca1144

• Gel Result Single band at length ~3000bp indicates successful digestion. Band was cut out and zymo purified.
  

Run stability test

• Per Template:DNA_Stability_Test_Protocol, begin at step 8.
  
• Run timepoints on a gel and determine whether degradation of DNA by endogenous nucleases occurred.
  
• 
  
  1. Ladder
  2. no plasmid
  3. 0 min
  4. 5 min
  5. 10 min
  6. 15 min
  7. 30 min
  8. 45 min
  9. 60 min
• Gel Result Based on the above gel, we know that the genomic DNA is being degraded over time. However, we do not see any indication of the plasmid on the gel, perhaps because the concentration added to each lane was too low. Future work would include adding more plasmid to the 1mL of lysate, or adding the 12uL of plasmid to much less lysate. Though the genomic DNA clearly degraded over time, we cannot extrapolate that information to the plasmid, since linearized plasmid may not degrade the same way as genomic DNA. Unfortunately we can not conclude anything about the plasmid stability in the self-lysis cell lysate from this gel.

Protocols

Test for DNA Stability to Endogenous Nuclease Activity
ZFN Enrichment Assay
phiC31 Assay Protocol