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Contents
September
September 2, 2010
ADS
Objective: Assemble lumazine-dT into pSB1C3 using NEB BioBrick Assembly Kit.
Method: Have the following parts: dT [28 ng/(µL)]; lumazine [78 ng/(µL)]; psB1C3 [14 ng/(µL)].
- Require 500 ng DNA each part in a 50(µL) rxn therefore 17.9(µL) dT and 6.4(µL) Lumazine. Need 250 ng pSB1C3 or 17.9(µL)
Restriction Reactions -
Ingredient Volume(µL) MilliQ H20 Water 36.1 NEBuffer 2 (10x) 5 Upstream Part (Lumazine) 26.4 EcoRI-HF 1 SpeI 1 100X BSA 0.5
Ingredient Volume(µL) MilliQ H20 Water 24.6 NEBuffer 2 (10x) 5 Downstream Part (dT) 17.9 XbaI 1 PstI 1 100X BSA 0.5
Ingredient Volume(µL) MilliQ H20 Water 3.35 NEBuffer 2 (10x) 2.5 Destination Plasmid (pSB1C3) 17.9 EcoRI-HF .5 PstI .5 100X BSA 0.25
- 2(µL) was removed prior to adding enzymes for analysis on gel.
- Split digestion reactions in half: dT - 2 @ 24(µL); Lum - 2 @ 24(µL); psB1C3 - 2 @ 11.5(µL)
- Ran 10 minute and 60 minute reactions at 37oC. Followed by 20 minute heat shock at 80oC.
- 2(µL) was removed from the heat killed reactions for analysis on gel.
- Performed 4 combinations of ligation: 10 min Restriction + 10 min Ligation; 10 min Restriction + Overnight Ligation; 60 min Restriction + 10 min Ligation; 60 min Restriction + Overnight Ligation
Ingredient 1X(µL) 2X Master Mix(x2.5)(µL) MilliQ H20 Water 11 77.5 T4 Ligase Buffer (10x) 2 5 Upstream Part 2 5 Downstream Part 2 5 T4 DNA Ligase 1 2.5 Plasmid Part 2 5 Incubated 10 minute and overnight ligations at room temperature ( 25oC). Heat killed ligase at 80oC for 20 min.
- 2(µL) of each reaction was taken for analysis on gel.
- Confirmed ligation with PCR analysis. Analyzed restriction, ligation and PCR on a 1.5% TAE agarose gel.
Component 1X(µL) Master Mix(x5.5)(µL) Milli-Q H2O 36.5 202.4 10x Pfu Buffer with MgSO4 5 27.5 dNTPs 2 11 Forward VF2 Primer 2 11 Reverse VR Primer 2 11 Template DNA 2 Pfu polymerase 0.2 1.1
Added 48(µL) to each reaction.
GEL PICTURE!!!
September 14, 2010
JF
Objective: Miniprep of RFP expression construct (J04450) via Qiaquick/Qiaprep spin column. Protocol followed as per kit instructions.
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Restriction Reactions -
Ingredient Volume(µL) MilliQ H20 Water 36.1 NEBuffer 2 (10x) 5 Plasmid DNA 26.4 EcoRI-HF 1 SpeI 1 100X BSA 0.5
Ingredient Volume(µL) MilliQ H20 Water 24.6 NEBuffer 2 (10x) 5 Downstream Part (dT) 17.9 XbaI 1 PstI 1 100X BSA 0.5
Ingredient Volume(µL) MilliQ H20 Water 3.35 NEBuffer 2 (10x) 2.5 Destination Plasmid (pSB1C3) 17.9 EcoRI-HF .5 PstI .5 100X BSA 0.25
- 2(µL) was removed prior to adding enzymes for analysis on gel.
- Split digestion reactions in half: dT - 2 @ 24(µL); Lum - 2 @ 24(µL); psB1C3 - 2 @ 11.5(µL)
- Ran 10 minute and 60 minute reactions at 37oC. Followed by 20 minute heat shock at 80oC.
- 2(µL) was removed from the heat killed reactions for analysis on gel.
- Performed 4 combinations of ligation: 10 min Restriction + 10 min Ligation; 10 min Restriction + Overnight Ligation; 60 min Restriction + 10 min Ligation; 60 min Restriction + Overnight Ligation
Ingredient 1X(µL) 2X Master Mix(x2.5)(µL) MilliQ H20 Water 11 77.5 T4 Ligase Buffer (10x) 2 5 Upstream Part 2 5 Downstream Part 2 5 T4 DNA Ligase 1 2.5 Plasmid Part 2 5 Incubated 10 minute and overnight ligations at room temperature ( 25oC). Heat killed ligase at 80oC for 20 min.
- 2(µL) of each reaction was taken for analysis on gel.
- Confirmed ligation with PCR analysis. Analyzed restriction, ligation and PCR on a 1.5% TAE agarose gel.
Component 1X(µL) Master Mix(x5.5)(µL) Milli-Q H2O 36.5 202.4 10x Pfu Buffer with MgSO4 5 27.5 dNTPs 2 11 Forward VF2 Primer 2 11 Reverse VR Primer 2 11 Template DNA 2 Pfu polymerase 0.2 1.1
Added 48(µL) to each reaction.
GEL PICTURE!!!
September 14, 2010
ADS
Objective: Assemble xylE-dT using 3AB assembly and 3AB assembly.
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 <partinfo>K118021</partinfo> 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 <partinfo>J04450</partinfo> 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) <partinfo>K331007</partinfo> - β-lactamase Bla Signal Sequence
- 2) <partinfo>K331008</partinfo> - Outer Membrane Protein ompA
- 3) <partinfo>K331009</partinfo> - Heat Stable Toxin I
- 4) <partinfo>K331012</partinfo> - Penicillin Binding Protein DacA
- All inserts in pMA-T vector (Standard Mr. Gene vector)
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 <partinfo>E1010</partinfo> 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.
- <partinfo>K331007</partinfo> (in pMA-T vector)
- <partinfo>K331008</partinfo> (in pMA-T vector)
- <partinfo>K331009</partinfo> (in pMA-T vector)
- <partinfo>K331012</partinfo> (in pMA-T vector)
Cells containing plasmids were put into glycerol stocks and put into HJ's -80oC.
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