Gibson Assembly Cloning of sfGPF into E. Coli
Introduction
Materials
- Software
Procedure
Order sfGFP Gene DNA - $158.05
- Create a new tube in transcriptic inventory, remember the code for the tube
micro-1.5
Store at -20C
no volume
- Go to IDT DNA
- Order 500ng of sfGFP from this IDT page
- Set the tube name in IDT DNA to be your code from transcriptic (here MFD)
- Ship to transcriptic (see how to set the address below)
Change accessioning <code> to match your accessioning code mentioned in the tube as "Please ship <code>" in transcriptic
- Wait for the DNA to arrive in your inventory (it took 4 business days / 6 calendar days to arrive)
- Validate: Confirm QC sequence in IDT order page (zip file with fasta file inside) matches sfGFP
Resuspend sfGFP DNA (link to python) - $0.46
- Centrifuge the tube for 3−5 sec at a minimum of 3000 x g to pellet the material to the bottom of the tube.
- Add 500µL TE (to reach 1ng/µL)
Adapted from IDT Fragment amplification instructions so that we have room for other uses for the DNA
- Briefly vortex and centrifuge
- Order via api (oligosynthesize)
- Centrifuge @ 2,000g for 30 secs
- Add 250µL TE to make 100µM solution of oligos
- Centrifuge @ 2,000g for 30 secs
- For each primer, ad 90µL of TE to a new tube
- For each primer, Add 10µL of the 100µM primer stock to the tube created in the previous step
Create Stock PCR Reagent Tubes (python) - $31.42
- Create pcr-plate with enough pcr reagent for 4 sets of 4 experiments
Q5 Polymerase: 14µL (we only need 8µL but we get up to 14µL for the same price b/c Q5 Buffer takes largest % of kit)
Q5 Buffer: 83µL
Q5 Enhancer: 83µL (not used in this protocol but comes basically free with kit)
10mM dNTP: 11µL
PCR of Insert (link to python) - $25.61
- Prep PCR plate for 4 experiments (1 per well)
A A | B B | C C | |
1 1 | Total uL | 25 | 100 |
2 2 | Q5 reaction buffer | 5 | 20 |
3 3 | Q5 polymerase | 0.25 | 1 |
4 4 | 10mM dNTP | 0.5 | 2 |
5 5 | 10uM forward primer | 1.25 | 5 |
6 6 | 10uM reverse primer | 1.25 | 5 |
7 7 | 1ng Template | 1 | 4 |
8 8 | Water | 15.75 | 63 |
3 have template (not 4)
1 is a negative control without template (well A2)
- Thermocycle
First 19 cycles are touchdown pcr
A A | B B | C C | |
1 1 | Recommended Ta | 64.8 | |
2 2 | Start Ta | 74.8 | |
3 3 | Stop Ta | 59.8 | |
4 4 | STEP | TEMP | TIME |
5 5 | Initial Denaturation | 98°C | 30 seconds |
6 6 | 15 Cycles | ||
7 7 | --melt | 98°C | 8 seconds |
8 8 | --anneal | 74.8°C -> 59.8°C (decrement by 1) | 25 seconds |
9 9 | --extend | 72°C | 8 seconds |
10 10 | 20 Cycles | ||
11 11 | --melt | 98°C | 8 seconds |
12 12 | --anneal | 59.8°C | 25 seconds |
13 13 | --extend | 72°C | 8 seconds |
14 14 | Final Extension | 72°C | 2 minutes |
15 15 | Hold | 4–10°C |
- Run gel
sample results:
- Enough for 8 experiments and dead volume
10µL of EcoRI
10µL of HindIII
48µL of Cutsmart Buffer 10x
- Setup 2 pcr wells with the following contents
A A | B B | C C | |
1 1 | Reagent | Volume per experiment (uL) | Contents |
2 2 | water | 42 | |
3 3 | pUC19 | 1 | 1ug DNA |
4 4 | CutSmart 10x | 5 | |
5 5 | EcoRI + HindIII | 2 | 20 units of each re |
6 6 | total volume | 50 |
- setup a third pcr well with the above contents except replace 2µL enzyme with 2µL water
- Run reaction: incubate for 15 minutes at 37C
- Inactivate Restriction Enzyme (RE): Thermocycle for 20 minutes at 80C
- Run Gel
sample results
- Consolidate
Run Gibson Assembly (python) - $21.45
- Put the following into a pcr well
A A | B B | C C | |
1 1 | Reagent | Volume (uL) | Notes |
2 2 | NEB Assembly Master Mix | 10 | |
3 3 | Vector | 2.5 | 0.067 pmols. This needs to be the limiting reagent since we don't want any left over that hasn't. 20 ng/ul * 3ul = 60ng. I lowered this from Brian's amount in order to create a higher ratio of insert without having to increase the volume of insert |
4 4 | Insert | 4 | .133 pmols Can't exceed 20% of reaction (4ul) 40 ng/ul * 4ul = 160ng |
5 5 | Water | 3.5 | |
6 6 | Total Volume | 20 |
- Thermocycle at 50C for 15 minutes
- Add 60µL water to the pcr well to dilute
Transform E. Coli (python) - $51.42
- Create two wells with the following reagents
CRITICALIn my experiment, I used 3µL of Gibson Assembly Product but 5µL would probably have been a better choice since we got very few colonies. The python has been updated for the new volume.
A A | B B | C C | |
1 1 | Reagent | Volume Experimental Well (uL) | Volume Control Well (uL) |
2 2 | Zymo DH5α | 50 | 50 |
3 3 | Gibson Assembly Product | 3 | 0 |
4 4 | pUC19 | 0 | 1 |
- Thermocycle:
5min @ 4C
30min @ 37C
- Add LB Broth
Mix 50µL of Experimental Well with 445µL of LB Broth in a tube
Mix 48µL of the Control Well with 207µL of LB Broth in a different tube
- Spread onto 6 plates with ampicillin and 6 without ampicillin in the following arrangement
A A | B B | C C | |
1 1 | 51uL Experimental | 57uL Experimental | 63uL Experimental |
2 2 | 69uL Experimental | 55uL Control | 65uL Control |
- Measure Growth by imaging each plate after 9 hours and again after 18 hours
Pick Colonies, Grow, and Measure Fluorescence (python) - $70.08 max (will be lower if there are fewer colonies than 50)
- Prepare 96-well pcr plate
A A | B B | C C | D D | |
1 1 | Reagent | Ampicillin Mix Volume (uL) | No Ampicillin Mix Volume (uL) | Notes |
2 2 | LB Miller | 1911 | 1913 | |
3 3 | Ampicillin | 2 | ||
4 4 | IPTG | 20 | 20 | required to free lac promoter site for transcription |
Add ampicillin mixture to 40 wells (Rows B/C/E/F, Cols 1-10)
Add no-ampicillin mix to 10 wells (Row D, Cols 1-10)
- Transfer 10 colonies from the 4 experimental flat plates (E. Coli containing assembled plasmid) and 10 colonies from 1 control plate (containing unmodified pUC19)
Expt Plate A1 --> PCR Plate Cols 1/2
Expt Plate A2 --> PCR Plate Cols 3/4
Expt Plate A3 --> PCR Plate Cols 5/6
Expt Plate B1 --> PCR Plate Cols 7/8
Control Plate B2 --> PCR Plate Cols 9/10
- Image Plates to confirm colonies have been picked
- Measure fluorescence and absorbance every 12 hours for 1 day
Flourescence:
excitation / emission
485nm / 535nm
Absorbance (to measure cell concentration): 600nm
- Wait for results:
Here are fluorescence results after 24 hours. It appears that only 24 hours of growth is all that is needed to reach maximal fluorescence.
- Store growth PCR plate at 4C
- Prepare two wells with 474.5µL LB + 0.5µL ampicillin in a larger growth container (96-deep)
- Mix/Transfer 25µL to each well from the highest fluorescing well
- Grow for 15 hours (until bacteria is in log phase of growth)
Incubate/shake at 37C
- Add 500µL of 40% glycerol to each well
- Store at -80C
This protocol will insert sfGFP into a pUC19 plasmid and then transform E.Coli using the new assembled plasmid. Its based on Brian Naughton's protocol. The total cost of the experiment was around $430.
The python for each section below can be checked using
python3 protocol.py | transcriptic analyze
Then ran via a test run
python3 protocol.py --test | transcriptic submit --project "Project Name" --title "my run" --test'
Then finally submitted for real (remove both --test parameters)
python3 protocol.py | transcriptic submit --project "Project Name" --title "my run"
Here is a link to the dna sequence of the assembled plasmid we will use for transformation (contains sfGFP flanked by EcorI and HindIII).