Team:Lethbridge/Notebook/Lab Work/September

Contents 1 September 20, 2010 1.1 ADS 2 September 21, 2010 2.1 ADS 3 September 21, 2010 3.1 ADS 4 September 23, 2010 4.1 JV 4.2 ADS 5 September 24, 2010 5.1 ADS

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 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

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