Team:Reading/Protocols

A note on protocols

Here we present a selection of the most important protocols we gathered over the course of our lab work. Many are adapted from freely available protocols and in these case a link is provided to the original. Any modifications we made are through trial and error experience on our part and may therefore not translate to your project. Acknowledgments are also listed thanking people who helped us with our project.

Contents

1. A Note on Protocols
2. Protocols
   • Miniprep
   • Glycerol stock
   • Optical Density
   • PCR
   • Transformation (E. coli)
   • Nanodrop
   • BG-11 plates
   • Cyanobacteria Transformation
   • Biobrick Assembly
3. References

Protocols

Isolation of plasmid DNA from bacteria (miniprep)

1. Pellet bacterial cells by centrifuging 1.5 ml of culture in a 1.5 ml microcentrifuge tube at 4000 rpm for 2 minutes
2. Discard supernatant by pipetting off ensuring not to disturb the pellet
3. Resuspend in 250 µl of resuspension solution by vortexing or pipetting up and down. Do not incubate for more than 5 minutes
4. Add 350 µl of neutralisation solution and mix by inverting the tube 4-6 times
5. Centrifuge at 13,000 rpm for 5 minutes
6. Transfer supernatant to a GeneJET spin column by pipetting. Do not disturb the white precipitate
7. Centrifuge the GeneJET spin column for 1 minute at 13,000 rpm
8. Discard the flow through
9. Add 500 µl of wash solution to the column
10. Centrifuge for 1 minute at 13,000 rpm
11. Discard flow through
12. Add 500 µl of wash solution to the column
13. Centrifuge for 1 minute at 13,000 rpm
14. Discard flow through
15. Centrifuge for 1 minute at 13,000 rpm
16. Transfer the GeneJET column to a new 1.5 ml microcentrifuge tube
17. Add 35 µl of ultrapure water. Do not touch the membrane with the pipette
18. Incubate at room temperature for 2 minutes
19. Centrifuge for 2 minutes at 13,000 rpm

Making glycerol stock

This protocol is adapted from 2 freely available protocols^{1, 2}. Ignore steps 2-4 if antibiotic was not present in the overnight broth. Work in a sterile cabinet

1. Take 0.5 ml from overnight culture and transfer to a centrifuge using sterile DNAase/RNAase free tips
2. Centrifuge at 13,000 rpm for 2 minutes
3. Discard supernatant
4. Add 0.5 ml of 60% glycerol stock
   1. 240 ml of glycerol
   2. 160 ml nano pure water
   3. mix together and autoclave
5. freeze at -80℃

Taking optical density of culture

Recommendation for taking OD for monitoring growth is either OD₇₃₀³ or OD₇₅₀⁴. Some recommend taking OD₇₃₀ at no higher than 0.4 because of problems with light scattering⁴. We chose to measure at growth OD₇₅₀ to keep in line with other high-profile papers on Synechocystis⁴.

1. Set spectrophotometer to measure at OD₇₅₀
2. Blank with 1 ml of BG-11 in a cuvette
3. Measure 1 ml of culture
4. If OD is over 1 dilute the 235 ul of culture in 750 ul of BG-11

Converting OD₇₅₀ to cell density For conversion of cell densities to numbers of cells, we have used the relationship OD750 = 1 (a.u) corresponding to 1.6 x 10⁸ cells mL⁵.

PCR

PCR was used as a means of amplifying each one of our biobrick constructs. Each construct, along with its corresponding flanking sequence of 50-100 bases, was amplified out of each transformed pSB1C3 plasmid.

Prior to PCR, it was ensured that all DNA obtained from miniprep was of a concentration of at least 1ng/ul per 100bp; this was performed using a desktop ThermoScientific NanoDrop machine. All PCR tubes were kept on ice prior to usage.

2μl of each of the VFR (forward) and VR (reverse) primers were added to sterile PCR tubes, along with 2μl of each respective transformed plasmid and 14μl phusion mastermix ⁶

PCR program program as follows:

1. Start:
   • 95℃ for 30 seconds
2. 35 cycles:
   • 95℃ for 10 seconds
   • 56℃ for 15 seconds
   • 72℃ for 70 seconds
3. Final
   • 72℃ for 5 minutes
   • 68℃ for 10 minutes

All PCR products were cleaned up using a ThermoScientific GeneJET PCR Purification Kit, using the provided protocol⁷

Transformation into E. coli

1. Thaw tubes of competent cells on ice and transfer 50 ul to a pre-chilled 1.5 ml Eppendorf
2. Add 5 ul of DNA using a sterile pipette tip
3. Flick the tubes to mix and then store on ice for 30 minutes
4. Heat shock in a water bath at 42℃ for 1 minute
5. Incubate on ice for 5 minutes
6. Add 450 ul of SOC (we used LB instead)
7. Place tubes horizontally at 37℃ for 2 hours on a shaker at 250 rpm
8. Invert Eppendorfs containing the cells several times
9. Plate out and incubate overnight at 37℃

Notes on transformation efficency

Expected transformation efficiency is 1 x 10⁶ cfu/ug of pUC19 DNA, but we should expect a 2-fold decrease in efficiency due to use of LB instead of SOC (a derivative of super optimal broth, SOB). We should also expect a decrease because we thawed the frozen competent cells at a temperature above 0ºC. Ideally they should be thawed on ice, or by hand if needed 8.

Nanodrop

Nanodrop is used to check concentration in DNA often from a miniprep

1. Set the Nanodrop to measure DNA
2. Blank the Nanodrop by placing 2 ul of PCR water on the stage and pressing blank
3. Add 2 ul of sample and measure. Measurement should be in ng/ul

BG-11 plates

BG-11 plates are used to grow Cyanobacteria. Antibiotics are added as needed for selection. 1.5% agar is used.

1. Add 7.55 g of agar to 500 ml BG-11 and autoclave to sterilise
2. If making kanamycin plates cool to ~55℃ and add 50 ug/ml
3. Agar melted in steamer and kept at 55℃ until needed for pouring

If making a kanamycin cap:

1. 0.6% agar w/v
2. Cool BG-11 to ~55ºC and add 0.5mg/ml kan
3. Add ~3ml to a plain BG-11 plate with transformed colonies on it
4. Leave for >1 week for Kan selection to occur

Cyanobacteria Transformation

Protocol to transform DNA into cyanobacteria

1. Make a fresh culture to OD₇₃₀=0.2 to 0.3 and grow for 3 days
2. Centrifuge 1.5 ml of culture at 4000g for 10 min. Remove supernatant
3. Repeat step 2
4. Add 1 ml BG-11, resuspend to wash, centrifuge at 4000g for 10 min, remove supernatant
5. Add 200ml fresh BG-11
6. 4ug plasmid A DNA is added (at a concentration of at least 100ng/ul)
7. Incubate for 24 under light on a shaker at ~100 rpm
8. Plate the full 200ml and leave for 1-2 days
9. Add 3-4ml kan 0.6% agar BG-11
10. Leave under light for >1 week

After many attempts to transform with Cyanobacteria we refined this protocol

1. Grow 6803 to ~0.435
2. Take 1 ml, re-wash with BG-11
3. Re-suspend in 90ml BG-11
4. Plasmid DNA at ~270ng/ul
5. add 8ul to each tube
6. place in 34ºC water bath, unshaken, in the dark, for 3 hours and 20 minutes
7. transfer to warm room at 28ºC and leave overnight
8. plate on Kan50 and leave under light, agar-side down

Biobrick Assembly

Protocol for inserting a construct into a plasmid backbone. In the case of BioBrick this will commonly be pSB1C3. Biobrick has 2 stages, a digestion stage and a ligation stage:

1. In a PCR tube add 20 ul of water and either 5 ul of your construct or 2 ul of plasmid backbone
2. To the same tube add 2.5 ul of NED buffer
3. Add appropriate restriction enzymes. In our case 1 ul of EcoR1 and 1 ul of Pst1
4. Incubate the tubes at 37℃ for 15 minutes
5. Then incubate at 80℃ for 20 minutes
6. Add 5 ul of water to a new tube followed by 2 ul of the digested construct and backbone
7. Add 2 ul of 10X T4 DNA ligase restriction buffer
8. Add 1 ul of T4 DNA ligase
9. Incubate at room temperature for 10 minutes
10. Incubate reaction mixture at 80℃ for 20 minutes. This step inactivates the enzyme

This DNA can now be used for transformation

References

1. Virginia Commonwealth University. To make Glycerol Stocks of Plasmids. [Online] Available at: http://www.people.vcu.edu/~pli/Protocols/Plasmid%20Preparation.pdf

2. OpenWetWare. 2012. Making a long term stock of bacteria. [Online] Available at: http://openwetware.org/wiki/Making_a_long_term_stock_of_bacteria

3. Eaton-Rye, J. J. in Photosynth. Res. Protoc. 295–312 (Humana Press, 2011). At . Accessed 20/08/2014.

4. Bradley, R. W., Bombelli, P., Lea-Smith, D. J. & Howe, C. J. Terminal oxidase mutants of the cyanobacterium Synechocystis sp. PCC 6803 show increased electrogenic activity in biological photo-voltaic systems. Phys. Chem. Chem. Phys. PCCP 15, 13611–13618 (2013).

5. Pojidaeva E, Zichenko V, Shestakov SV, Sokolenko A (2004) Involvement of the SppA1 peptidase in acclimation to saturating light intensities in Synechocystis sp. strain PCC 6803. J Bacteriol 186: 3991–3999.

6. Pojidaeva E, Zichenko V, Shestakov SV, Sokolenko A (2004) Involvement of the SppA1 peptidase in acclimation to saturating light intensities in Synechocystis sp. strain PCC 6803. J Bacteriol 186: 3991–3999.

7. ThermoScientific. 2013. Thermo Scientific GeneJET PCR Purification Kit #K0701, #K0702. [Online] Available at: http://www.thermoscientificbio.com/uploadedFiles/Resources/k070-product-information.pdf

8. New England BioLabs. 2014. High Efficiency Transformation Protocol (C2987H/C2987I). [Online] Available at: https://www.neb.com/protocols/1/01/01/high-efficiency-transformation-protocol-c2987

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