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
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.
July 17th
Goal
to extract plasmid of PB19 from the transformed cells.
Procedure
1. Growth the cell overnight
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 mini prep)
Results
Tube 1: 40 ng/ul
Tube 2: 30ng/ul
Goal
to digest the segment using AgeI and XbaI. Purify the digested product.
Procedure
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
Concentration after purification: 12.2 ng/ul
July 23rd
Goal
: to achieve the great great goal of magnifying the RBS and promoter before digestion, purification and ligation.
Procedure
we did a nano drop of the mini prep plasmid, and it is 460ng/ul. The curve looks good. We diluted 1:500
Results
a fainted band was seen. However, the concentration after purification is below 5 ng/ul, too low to be used to ligation. Therefore, we decided to design the sequence as two oligos, with Xbal and Agel sticky ends.
August
August 4th
Goal
to dilute received oilgos, to Phosphorylate them, to Anneal them and to ligation is with Limonene vector
Procedure
1. Dilute the oilgo to 100 uM, the stock
2. Take 10 ul out of the 100 uM stock and dilute to make 10 uM working stock
3. Mix:
2 uL 10 µM sense oligo
2 uL 10 µM anti-sense oligo
2 uL 10 x PNK (polynucleotide kinase) buffer A
2 uL 10mM ATP
1 uL T4 polynucleotide kinase (PNK)
10 uL distilled water
to give 20 uL total volume
4. Incubate at 37C for 30 mins
5. Place in boiling water bath for 2 min, then remove water bath from the heat source and allow the reaction (still in the water bath) to cool to room temperature.
(Labelled Annealing in the box)
6. ligation
Mix
8 ul of vector (12 ng/ul)
1 ul of insert
0.5 ul of ligase enzyme
2 ul of ligase buffer
8.5 ul of distilled water
total 20 ul
a. put under 22 degree for 30 minutes
b. put under 16 degree for 30 minutes
c. put in 4 degree overnight
Results
colonies were seen after transforming and plating.
August 10th
Goal
to sequence the ligated plasmid for limonene construct
Procedure
Dilute the mini prep product 1: 2 (Give us concentration 70ng/ul and 80ng/ul)
design primers that are about 200 bp away upstream and downstream from the inserted sequence
Send them to GATC
Results
It appears that the old plasmid recirculate itself.
Further Steps
regrow 6 more colonies, minipreped and did analytical PCR with NotI and AgeI
Colony 1a appears to have the complete plasmid.
August 20th
Goal
to PCR ilvBN gene from MG1655
Procedure
1. Picked 2 colonies from the plate MG1655 115+GFP
2. inoculate them each in 50 ul of NF H2O
3. Boiled for 3 mins at 98 degree
4. use the solution as DNA template
5. follow standard PCR protocol from this step on
| Reagent | Volume |
| 1x |
| Nuclease-free water | 63 ul |
| 5x Phusion HF Buffer | 20 ul |
| 10 mM dNTPs | 2 ul |
| Forward Primer (10 uM) | 5 ul |
| Reverse Primer (10 uM) | 5 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 min | extend |
| finish | 72 C | 5 min | extend |
| blind | 10 C | forever | blind |
Results
Band seen on both sample (from 2 colonies)
August 21st
Goal
to Digest the PCR product (ilvBN) from yesterday and to digest pET-DUET1 as vector
Procedure
Vector (pET-DUET1)
| Reagent | Volume |
| Nuclease-free water | 38 ul |
| 10x FD Buffer | 5 ul |
| DNA | 2 ul |
| EcoRI | 2 ul |
| NcoI | 2 ul |
| FastAP | 1 ul |
| Total Volume | 50 ul |
ilvBN
| Reagent | Volume |
| Nuclease-free water | 39 ul |
| 10x FD Buffer | 5 ul |
| DNA | 2 ul |
| EcoRI | 2 ul |
| NcoI | 2 ul |
| Total Volume | 50 ul |
Incubate at 37 degree for 2 hours. Purification using the PCR purification kit.
Results
24 ng/ul for C2 and 12 ng/ul for C1
(Will use C2 PCR purified product for digestion.)
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