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.
July
July 1st
Goal
to design the primers and gBlock to modify the BioBricks part BBa_I742111.
Procedure
1. decide the major steps of modification
a. to PCR the biobricks construct using primers that have Agel restriction site sequences.
b. to digest the PCR product
c. to PCR the gBlock with Promoter, RBS and restriction sites (Xbal and Agel)
d. to digest the PCR product
e. to ligate the two sequences together
2. Design the primers for PCR and the gBlock including promoter, RBS and restriction sites using the software Geneious.
Results
fwd primer for BioBricks part PCR: 0PB.015
rev primer for BioBricks: 0PB.016
gBlock with promoter, RBS and restriction sites: 0PB. 019
fwd primer for gBlock PCR: 0PB.30
rev primer for gBlock PCR: 0PB.31
(We ended up ordering the gBlock as synthetic gene sequence, because it is too long for oligos and too short for gBlocks. Oh well.)
July 7th
Goal
to extract plasmid of biobricks I742111 from the transformed cells.
Procedure
1. Growth the cell for two days
2. Centrifuge the tube for 15 minutes with 4000 rps
3. Follow the standard protocol of Thermo Scientific miniprep kit
4. Label the centrifuge tube and store it in the -20 freezer. (Syl miniprep)
Results
1. We grew two tubes of glycerol stocks. However living cells were only seen in one of them, which was used for miniprep.
2. Plasmid stored in the box after the procedure. PCR would be done tomorrow.
July 8th
Goal
Amplify limonene synthase gene sequence for E.coli
Fw primer: oPB.015
Rv primer: oPB.016
Procedure
| Reagent | Volume |
| 1x |
| Nuclease-free water | 71 ul |
| 5x Phusion HF Buffer | 20 ul |
| 10 mM dNTPs | 2 ul |
| Forward Primer (10 uM) | 1 ul |
| Reverse Primer (10 uM) | 1 ul |
| Template Plasmid | 1 ul |
| Phusion DNA Polymerase | 1 ul |
| DMSO | 3 ul |
| Total Volume | 100 ul |
> | Temperature | Time | >Function |
| start | 98 C | 30 sec | melt |
| cycle 1 | 98 C | 10 sec | melt |
| cycle 2 | 51 C | 30 sec | anneal |
| cycle 3 | 72 C | 30 sec | extend |
| finish | 72 C | 5 min | extend |
| blind | 10 C | forever | blind |
Results
PCR product at the expected length.
July 9th
Goal
I. to run the Gel of the PCR product to confirm that the segment is at the right length.
II. to digest the segment using AgeI and XbaI. Purify the digested product.
Procedure
I. a. take the PCR tubes out of the machine.
b. Mixing the dye with PCR products, from each tube.
c. Run the gel for an hour and half using 100bp plus DNA ruler
d. Observe under UV light
II. a. digest in the 37 degree incubator for 10 minutes using AgeI and XbaI.
| Reagent | Volume |
| Nuclease-free water | 27.5 ul |
| 10x FD Buffer | 5 ul |
| DNA | 12.5 ul |
| AgeI | 2.5 ul |
| Xbal | 2.5 ul |
| Total Volume | 50 ul |
b. Purify following the protocol of digestion purification kit. Used warm NF water for diluting DNA at the last step.
Results
The PCR product is at expected length.
Purified product has a very low concentration(2.5ng/ul), meaning the PCR product was a mixture of multiple segments and didn't give ideal result. Therefore proceed to PCR again.
July 16th
Goal
Amplify limonene synthase gene sequence for E.coli
Fw primer: oPB.015
Rv primer: oPB.016
Procedure
| Reagent | Volume |
| 1x |
| Nuclease-free water | 71 ul |
| 5x Phusion HF Buffer | 20 ul |
| 10 mM dNTPs | 2 ul |
| Forward Primer (10 uM) | 1 ul |
| Reverse Primer (10 uM) | 1 ul |
| Template Plasmid | 1 ul |
| Phusion DNA Polymerase | 1 ul |
| DMSO | 3 ul |
| Total Volume | 100 ul |
> | Temperature | Time | >Function |
| start | 98 C | 30 sec | melt |
| cycle 1 | 98 C | 10 sec | melt |
| cycle 2 | 51 C | 30 sec | anneal |
| cycle 3 | 72 C | 2.5 min | extend |
| finish | 72 C | 5 min | extend |
| blind | 10 C | forever | blind |
Results
It worked!
Goal
: transform the PB19 synthase part into e.coli.
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
Colonies seen on both plates.
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