Team:Lethbridge/Notebook/Lab Work/September

From 2010.igem.org

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((ADS))
((ADS))
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<b>Results:</b> TBD <br>
<b>Results:</b> TBD <br>
<b>Follow-up:</b> TBD<br>
<b>Follow-up:</b> TBD<br>
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<b>Objective:</b> Create glycerol stocks of J04450 in pSB1A3, pSB1K3 and pSB1T3 for use in RFP-BioBrick Assembly.<br>
<b>Objective:</b> Create glycerol stocks of J04450 in pSB1A3, pSB1K3 and pSB1T3 for use in RFP-BioBrick Assembly.<br>
<b>Method:</b> Transform into Subcloning Efficiency Competent DH5&alpha; Cells (Invitrogen)<br>
<b>Method:</b> Transform into Subcloning Efficiency Competent DH5&alpha; Cells (Invitrogen)<br>
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*Generate Glycerol Stocks
*Generate Glycerol Stocks
*Generate Plasmid DNA via Maxiprep
*Generate Plasmid DNA via Maxiprep
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<b>Objective:</b> Create glycerol stocks of received synthesized (Mr. Gene) signal peptides.
<b>Objective:</b> Create glycerol stocks of received synthesized (Mr. Gene) signal peptides.
<b>Method:</b> Transform into Subcloning Efficiency Competent DH5&alpha; Cells (Invitrogen) plasmid DNA containing the following BioBricks:
<b>Method:</b> Transform into Subcloning Efficiency Competent DH5&alpha; Cells (Invitrogen) plasmid DNA containing the following BioBricks:

Revision as of 03:32, 25 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 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 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)

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, pSB1K3 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 pSB1K3 - Well 5A
  • 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