Team:Paris Bettencourt/Notebook/Odor Library

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Odor Library



Notebook

June

June 26th
Goal

Design plasmid for limonene synthase

Procedure

Used MG1665 strain, the RBS with the highest initial rate and the sequence of r-limonene synthase from genBank. Added polyhistidine tail to the construct. Submitted the construct to Jake.

Results

Designed plasmid for limonene synthase using the software geneious. Found a biobricks part containing this sequence. For the purpose of saving budget, we would first transform this standard part into cell and later modify it.

June 27th
Goal

: transform the bioBricks limonene synthase(BBa_I742111) part into e.coli.

  • We found that the bioBricks kit for 2014 contain the limonene synthase sequence in plate 4 position 3I.

    • Procedure

  • Start thawing the competent cells(Used Christina's competent cells) on ice.
  • Add 50 µL of thawed competent cells into pre-chilled 2ml tube, and another 50µL into a 2ml tube, labelled for your control.
  • Add 2 µL of the resuspended DNA to the 2ml tube. Pipet up and down a few times, gently. Make sure to keep the competent cells on ice.
  • Close tubes and incubate the cells on ice for 30 minutes.
  • Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds.
  • Incubate the cells on ice for 5 minutes.
  • Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking. Important: 2 hour recovery time helps in transformation efficiency, especially for plasmid backbones with antibiotic resistance other than ampicillin.
  • Label two petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid backbone, and antibiotic resistance. Plate 20 µl and 200 µl of the transformation onto the dishes, and spread. This helps ensure that you will be able to pick out a single colony.
  • For the control, label two petri dishes with LB agar (CM). Plate 20 µl and 200 µl of the transformation onto the dishes, and spread.
  • You can pick a single colony, make a glycerol stock, grow up a cell culture and miniprep.

    • Results

    transformed the plasmid containing limonene synthase sequence from biobricks into E.Coli. Colonies seen on both plates. Glycerol Stock made: PB. 018 and PB. 019

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    July

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    August

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    September

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    October

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