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Contents
September
September 20, 2010
ADS
Objective: Analyze PCR of mms6 (HB) and fluorescent proteins (AV).
Method: Analyzed on 2% TAE agarose gel.
Load order:
Lane Sample Volume
Sample (µL)Volume Loading
Dye (µL)1 HB1 10 2 2 HB2 10 2 3 HB3 10 2 4 HB4 10 2 5 HB5 10 2 6 100bp Ladder (Fermentas) 0.5 2(+10 H20) 7 RFP1 10 2 8 RFP2 10 2 9 RFP3 10 2
Results: ADD IMAGE
- mms6 was not amplified
- RFP was amplified
- Expected size is ~800bp
- Actual size is >1000bp
- We believe that the suffix segment of the fusion primer annealed rather than the FP segment. This would cause the promoter (R0040) of J04450 to be amplified, adding ~200bp, giving a final size of >1000bp.
Objective: Confirm assembly (3 antibiotic) of K118021 and B0015 from Sept. XX, 2010 via PCR.
Method: Set up 50uL reaction mixture with 2uL of ligated DNA. Used VF2 and VR primers.
Used PFU setting on Thermocycler
Results: ADD IMAGE
Conclusion: Assembly did not work as intended.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 21, 2010
ADS
Objective: Analyze PCR of K118021-B0015 and subsequent PCR (ADS) and PCR of K118021 to add either N or C terminal fusion standard (KG).
Method: Analyze on 2% TAE agarose gel.
Load Order:
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)
- Incubated 30 min at RT
- 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
September 24, 2010
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
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