Team:Lethbridge/Notebook/Lab Work/September

Contents 1 September 21, 2010 2 September 23, 2010 2.1 (JV) 2.2 (ADS)

September 21, 2010

===(ADS)===
Objective: Insert xylE (with N and C terminal fusion standards, obtained by PCR of K118021 by KG) into pSB1C3 plasmid for submission to registry.

Method:

• Restriction of xylE PCR product and pSB1C3 (containing J04450 biobrick) via BioBrick Method using EcoRI-HF and PstI (Enzymes from NEB)
• Ligation of xylE PCR product and pSB1C3 via BioBrick Method using T4 DNA ligase (Enzyme from NEB)
• Transform into Library Efficiency Compentent DH5α Cells (Invitrogen)

Results:

• Obtained too numerous to count (TNTC) colonies.
  • Obtained ~100 white colonies (indicating removal of RFP and insertion of new BioBrick)

Note:
Parent plasmid (from PCR) not digested, possibly K118021 moved into pSB1C3 backbone.

Follow-up:
Screen white colonies by addition of catechol to solution containing white cells.

September 23, 2010

(JV)

Objective: Characterized catechol degradation by xylE enzyme

Method: Measured absorbance of catechol (275nm) and 2-hydroxymuconate semialdehyde (380nm).

• Protocol:
• 1) Grow cells in M9 minimal medium
• 2) Take 1/10 dilution of cells
• 3) Introduce 1µL of 0.05M catechol solution into the cell dilution. (Final concentration of 50&microM;).
• 4) Quench the reaction with 5A% w/v trichloroacetate at certain time points. (0,15sec, 30sec, 45sec, 60sec, 2min, 3min, 4min, 5min, 10min).
• 5) Spin down cells.
• 6) Measure absorbance of supernatant.
  

Results:

(ADS)

Objective: Move xylE (two biobrick; one with Fusion prefix, one with fusion suffix) into pSB1C3.
Method:

• Restriction of xylE PCR product and pSB1C3 (containing J04450 biobrick) via BioBrick Method using EcoRI-HF and PstI (Enzymes from NEB)
• Ligation of xylE PCR product and pSB1C3 via BioBrick Method using T4 DNA ligase (Enzyme from NEB)
  • Incubated 30 min at RT
• Transform into Subcloning Efficiency Compentent DH5α Cells (Invitrogen)

Results: TBD
Follow-up: TBD

Objective: Create glycerol stocks of received synthesized (Mr. Gene) signal peptides. Method: Transform into Subcloning Efficiency Competent DH5α Cells (Invitrogen) plasmid DNA containing the following BioBricks:

• 1) K331007 - β-lactamase Bla Signal Sequence
• 2) K331008 - Outer Membrane Protein ompA
• 3) K331009 - Heat Stable Toxin I
• 4) K331012 - Penicillin Binding Protein DacA

Results: Obtained TNTC Cells
Follow-up:

• 1) Grow overnight cultures
• 2) Purify pDNA
• 3) Move into pSB1C3 plasmid
• 4) Verify sequence
• 5) Submit to registry for sequencing