Team:Lethbridge/Notebook/Lab Work/September

From 2010.igem.org

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<b>Method:</b>Qiagen spin column protocol.<br>
<b>Method:</b>Qiagen spin column protocol.<br>
-
*K331007 (in pMA-T vector)
+
*<partinfo>K331007</partinfo> (in pMA-T vector)
-
*K331008 (in pMA-T vector)
+
*<partinfo>K331008</partinfo> (in pMA-T vector)
-
*K331009 (in pMA-T vector)
+
*<partinfo>K331009</partinfo> (in pMA-T vector)
-
*K331012 (in pMA-T vector)<br>
+
*<partinfo>K331012</partinfo> (in pMA-T vector)<br>
Cells containing plasmids were put into glycerol stocks and put into HJ's -80<sup>o</sup>C.<br>
Cells containing plasmids were put into glycerol stocks and put into HJ's -80<sup>o</sup>C.<br>

Revision as of 03:30, 26 September 2010



Feel free to look around our notebook!

Here you can check out the work we have done in the lab, click on a month to take a look!

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:

LaneSampleVolume
Sample (µL)		Volume Loading
Dye (µL)
1HB1102
2HB2102
3HB3102
4HB4102
5HB5102
6100bp Ladder (Fermentas)0.52(+10 H20)
7RFP1102
8RFP2102
9RFP3102

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:

LaneSampleVolume
Sample (µL)		Volume Loading
Dye (µL)
1xylE F-S 151
2xylE F-S 251
3xylE F-S 351
4xylE F-S 451
5xylE F-S 551
6xylE F-S 651
7100bp Ladder (NEB)0.51(+5 H20)
8xylE S-F 151
9xylE S-F 251
10xylE S-F 351
11xylE S-F 451
12xylE S-F 551
13xylE S-F 651
14Empty
15PCR of K118021-B0015 (ADS)11
16Empty
17Empty

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: 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 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.

  • <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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