| Lane | Sample | Volume
Sample (µL) | Volume Loading
Dye (µL) |
| 1 | xylE F-S 1 | 5 | 1 |
| 2 | xylE F-S 2 | 5 | 1 |
| 3 | xylE F-S 3 | 5 | 1 |
| 4 | xylE F-S 4 | 5 | 1 |
| 5 | xylE F-S 5 | 5 | 1 |
| 6 | xylE F-S 6 | 5 | 1 |
| 7 | 100bp Ladder (NEB) | 0.5 | 1(+5 H20) |
| 8 | xylE S-F 1 | 5 | 1 |
| 9 | xylE S-F 2 | 5 | 1 |
| 10 | xylE S-F 3 | 5 | 1 |
| 11 | xylE S-F 4 | 5 | 1 |
| 12 | xylE S-F 5 | 5 | 1 |
| 13 | xylE S-F 6 | 5 | 1 |
| 14 | Empty | | |
| 15 | PCR of K118021-B0015 (ADS) | 1 | 1 |
| 16 | Empty | | |
| 17 | Empty | | |
Results: ADD IMAGE
- Both KG PCR (Standard-Fusion and Fusion-Standard) amplified
- ADS PCR Amplified, but no insert present.
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 50µM;).
- 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: Cuvette used interfered with Spectra.
ADS
NOTE: In all transformations, heat shock step was missed. HOWEVER, all transformations showed significant number of colony forming units.
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)
- Transform into Subcloning Efficiency Compentent DH5α Cells (Invitrogen)
Results: TBD
Follow-up: TBD
Objective: Create glycerol stocks of J04450 in pSB1A3 and pSB1T3 for use in RFP-BioBrick Assembly.
Method: Transform into Subcloning Efficiency Competent DH5α Cells (Invitrogen)
Obtained all plasmid DNA from 2010 Kit Plate 1
- J04450 in pSB1A3 - Well 1C
- J04450 in pSB1T3 - Well 7A
Results: Obtained TNTC colonies
Follow-up:
- Grow overnight cultures
- Generate Glycerol Stocks
- Generate Plasmid DNA via Maxiprep
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
ADS
Objective: Generate plasmid DNA of E1010 for downstream PCR
Method: Transform plasmid DNA into Subcloning Efficiency Competent DH5α Cells (Invitrogen)
DNA obtained from 2010 Kit Plate 1 Well 18F (E1010 in pSB2K3)
Results: Obtained TBD colonies
Follow-up:
- Grow overnight cultures (Generate glycerol stocks)
- Purify plasmid DNA (Generate pDNA stocks)
- PCR to add terminal fusion standards
Objective: Create glycerol stocks of J04450 in pSB1K3 for use in RFP-BioBrick Assembly.
Method: Transform into Subcloning Efficiency Competent DH5α Cells (Invitrogen)
Obtained plasmid DNA from 2010 Kit Plate 1 well 5A (J04450 in pSB1K3)
Results: Obtained TNTC colonies
Follow-up:
- Grow overnight cultures
- Generate Glycerol Stocks
- Generate Plasmid DNA via Maxiprep
September 25, 2010
JV
Objective: Extract Plasmid DNA from DH5α cells.
Method:Qiagen spin column protocol.
- K331007
- K331008
- K331009
- K331012
Cells containing plasmids were put into glycerol stocks and put into HJ's -80oC.
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