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
June 28th
Goal
Made chemical competent cell following standard protocol with MG1665 strains.
Procedure
1. The night before, inoculate a 5 ml culture and grow overnight with selection.
2. The day of the experiment dilute cells ~ 1:200 into selective media.
For this example add 250 ul to 50 ml of selective media.
Note: The protocol is easily scaled to increase the number of cells.
3. Grow the cells to an OD600 of 0.6 – 0.7.
Use a large flask, 500ml, for good aeration.
Use a baffled flask for fastest growth.
This takes about 3 hours depending on the cells.
Medium-heavy cloudiness by eye is fine.
4. Spin down the cells at 4 ºC, 4000 rpm, 15 minutes. Note: Keep the cells at 4 ºC from now on.
5. Resuspend cells in 15 ml, ice-cold 100 mM CaCl2. Leave on ice 4 hours to overnight.
6. Spin down the cells at 4 ºC, 4000 rpm, 15 minutes.
7. Resuspend cells in 4 ml, ice-cold 100 mM CaCl2 + 15% glycerol.
8. Aliquot into pre-chilled Eppendorf tubes. Use immediately or store at -80ºC.
Note: Frozen cells are only good once.Do not refreeze cells once thawed.
Results
The transformed cell were grown for 20 hours and the transformation was successful. The plates were put into the 4 degree fridge for making stocks on Monday.
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