Team:HKJS S/Experiments
Experiment
Protocols
Inoue Method for Preparation of “Ultra-competent” Cells
1. Prepare Inoue transformation buffer
2. Streak out bacteria from freezer stock on LB-agar plate and grow overnight at 37°C
3. Pick a single colony and transfer to 25 mL of LB media in a 250 mL flask
4. Incubate culture for 6 – 8 hours at 37°C with vigorous shaking
5. At ~6 pm, inoculate 3 1-liter flasks, each with 250 mL of LB media
a. 1st flask = 10 mL of starter culture
b. 2nd flask = 4 mL of starter culture
c. 3rd flask = 2 mL of starter culture
6. Incubate 3 flasks overnight at 18°C
7. The following morning, take OD600 of the three flasks.
8. Monitor the flasks until one flask reaches OD600 = 0.55
9. Transfer this flask to an ice bath for 10 minutes and discard the other 2 cultures
10. Centrifuge cells at 2500g (~3500 rpm in sorvall) for 10 minutes at 4°C
11. Remove all media from samples. Use an aspirator if possible
12. Resuspend cells in 80 mL of ice-cold Inoue transformation buffer
13. Spin cells again at 2500g for 10 minutes at 4°C
14. Remove all buffer
15. Add 20 mL of ice-cold Inoue transformation buffer
16. Add 1.5 mL of DMSO
17. Mix the bacterial suspension by swirling and then store it on ice for 10 minutes
18. Dispense aliquots into eppendorf tubes and snap freeze in MeOH/dry ice
19. Store at -70°C
2. Streak out bacteria from freezer stock on LB-agar plate and grow overnight at 37°C
3. Pick a single colony and transfer to 25 mL of LB media in a 250 mL flask
4. Incubate culture for 6 – 8 hours at 37°C with vigorous shaking
5. At ~6 pm, inoculate 3 1-liter flasks, each with 250 mL of LB media
a. 1st flask = 10 mL of starter culture
b. 2nd flask = 4 mL of starter culture
c. 3rd flask = 2 mL of starter culture
6. Incubate 3 flasks overnight at 18°C
7. The following morning, take OD600 of the three flasks.
8. Monitor the flasks until one flask reaches OD600 = 0.55
9. Transfer this flask to an ice bath for 10 minutes and discard the other 2 cultures
10. Centrifuge cells at 2500g (~3500 rpm in sorvall) for 10 minutes at 4°C
11. Remove all media from samples. Use an aspirator if possible
12. Resuspend cells in 80 mL of ice-cold Inoue transformation buffer
13. Spin cells again at 2500g for 10 minutes at 4°C
14. Remove all buffer
15. Add 20 mL of ice-cold Inoue transformation buffer
16. Add 1.5 mL of DMSO
17. Mix the bacterial suspension by swirling and then store it on ice for 10 minutes
18. Dispense aliquots into eppendorf tubes and snap freeze in MeOH/dry ice
19. Store at -70°C
| Inoue transformation buffer | 1 liter |
| 55 mM MnCl2 | 10.88 g |
| 15 mM CaCl2 | 2.20 g |
| 250 mM KCl | 18.65 g |
| 10 mM PIPES (0.5 M, pH 6.7) | 20 mL |
| H2O | To 1 L |
PIPES
- 15.1 g PIPES in 80 mL of H2O
- Adjust to pH 6.7 with 5M KOH and then bring to 100 mL Sterilize by filtration
- Store aliquots at -20°C
- 15.1 g PIPES in 80 mL of H2O
- Adjust to pH 6.7 with 5M KOH and then bring to 100 mL Sterilize by filtration
- Store aliquots at -20°C
Gibson Assembly
1. Two or more gBlocks Gene Fragments are designed with 20–80 base overlaps with the adjacent gBlocks fragment sequences and the linearized plasmid. The plasmid can be linearized by restriction digest or PCR.
2. Linearized plasmid and fragments are combined in a tube with Gibson Assembly® Master Mix (New England Biolabs).
gBlocks® Gene Fragments and Plasmid Assembly
1Use 50–100 ng of plasmid and a 2–3 fold excess of insert fragments. Table source: www.NEB.com/GibsonAssembly.
3. Incubate at 50oC for 1 hr.
4. The resulting completed plasmid is ready for transformation into bacteria, and analysis.
2. Linearized plasmid and fragments are combined in a tube with Gibson Assembly® Master Mix (New England Biolabs).
gBlocks® Gene Fragments and Plasmid Assembly
| Total # of fragments including plasmid | 2–3 Fragments | 4–6 Fragments |
| Quantity1 | 0.02–0.50 pmole ea. | 0.05–5.0 pmole ea. |
| Gibson Assembly® Master Mix (2X) | 10 μL | 10 μL |
| Deionized H2O | Adjust to final 20 μL | Adjust to final 20 μL |
| Total volume | 20 μL | 20 μL |
3. Incubate at 50oC for 1 hr.
4. The resulting completed plasmid is ready for transformation into bacteria, and analysis.
Experiment Methods
Gene synthesis
The gene cluster which enables the carbon dioxide reduction is synthesised according to nif genes of Paenibacillus polymxya WLY 78, with amino acid substitutions of V70A and H195Q. The gene of interest is then silent mutated by editing the file to remove all restriction sites EcoRI, XbaI, SpeI, PstI, as to fulfil biobrick standards. Furthermore, biobrick prefix and suffix are added before and after the sequence. The resulted sequence is stated in the Supporting Information.
The gene of interest is synthesised with Integrated DNA Technologies (IDT) gblocks® Gene Fragments (gblocks®), a service that provides linear, double-stranded and sequenced DNA fragments [1]. As the length of gblocks® can only be up to 3000 bp [1], while nif genes are 10.9 kb in length, the ordering was separated into four parts, 2187, 2187, 2187 and 2389 in length respectively, with 50 bp overlapping region.
[1] Integrated DNA Technologies, “gBlocks® Gene Fragments,” [Online]. Available: http://sg.idtdna.com/pages/products/genes-and-gene-fragments/gblocks-gene-fragments. [Accessed 26 May 2018].The gene of interest is synthesised with Integrated DNA Technologies (IDT) gblocks® Gene Fragments (gblocks®), a service that provides linear, double-stranded and sequenced DNA fragments [1]. As the length of gblocks® can only be up to 3000 bp [1], while nif genes are 10.9 kb in length, the ordering was separated into four parts, 2187, 2187, 2187 and 2389 in length respectively, with 50 bp overlapping region.
Gene resuspension
Upon the arrival of the gene fragments, the pellets are processed according to the instructions given by the manufacturer [2]. The pellets containing genes are first centrifugated 3-5 seconds with 3000x g to pellet the material to the bottom of the tube. Next, 20 µL TE is added to have a final concentration of 50 ng/µL, then vortexed and centrifuged briefly. The final processed genes are resuspended in in nuclease-free water.
[2] Integrated DNA Technologies, “gBlocks™ Gene Fragments Cloning Protocols,” [Online]. Available: http://sfvideo.blob.core.windows.net/sitefinity/docs/default-source/user-guide-manual/gblocks-gene-fragments-protocols.pdf?sfvrsn=1c133407_6. [Accessed 26 May 2018].Assembly of gene cluster
The genes will be based on the assembly method proposed by Danial Gibson et. al. [3]. In the overlapping regions, a T5 exonuclease cleaves the 5' end of each fragment, resulting in overhangs, then the exonuclease is deactivated at 50ºC. This results in the annealing of overlapping fragments, hence a complete DNA cluster. Next, a DNA polymerase and ligase next fill in gaps and nicked parts in the gene of interest respectively.
Firstly, fragment 1 and 5 will be ligated to the linearized plasmid, then fragments 2, 3, 4 will be assembly by Gibson assembly. This results in two fragments containing the plasmid in one of them. Lastly, a Gibson assembly will be performed and can be directly used in transformation.
[3] D. G. Gibson, L. Young, R.-Y. Chuang, J. C. Venter, H. I. Clyde A and H. O. Smith, “Enzymatic assembly of DNA molecules up to several hundred kilobases.,” Nature Methods, vol. 6, pp. 343-345, 2009.Firstly, fragment 1 and 5 will be ligated to the linearized plasmid, then fragments 2, 3, 4 will be assembly by Gibson assembly. This results in two fragments containing the plasmid in one of them. Lastly, a Gibson assembly will be performed and can be directly used in transformation.
Transformation
The transformation will be done by Heat-shock method by the protocol provided by igem.
Selection
When transformed using pET-blue-2 as the plasmid backbone, a blue-white screen can be used to screen the colonies. The colony will be grown overnight in 2 mL of LB solution then the plasmid can be miniprepped, and subject to a gel electrophoresis screen.