User:Chris D Hirst/Protocols

Protocols

Here will be placed any finished or currently worked on protocols until they are deemed of a high enough standard to be put on the Imperial iGEM 08 wetware wiki.

Cell Count v Optical Density Curve Calibration

Aim

To produce a calibration curve to aid in the normalising of fluorescence values to allow proper characterisation of Promoters and RBSs for B.subitlis. This protocol must give results that are as accurate as possible over a considerable range of Optical Densities.

Equipment

Spectrophotometer

Cuvettes

P20, P200 and P1000 Gilsons

50mL flask (an additional one for each repeat)

Timer

Reagents

LB Medium

Lots of LB Agar plates

Protocol

Be VERY careful to avoid contamination at all stages

1. Grow up a culture of B.subtilis overnight
2. Prepare a 50mL flask of LB medium, adding the required antibiotics (if any) and take 1mL to use as a blank
3. Pipette 500μL into the 50mL of LB medium and mix thoroughly
4. Immediately take 1mL of the new culture and measure the OD against the blank
   1. Dilute 100μL of culture in 900μL of LB and mix well
   2. Plate 100μL of this diluted solution
   3. Repeat the dilution and plating 8 times to obtain plates for colony counting
      1. Be sure to mark the OD, time and dilution for each plate!
5. At 60 minutes, 90 minutes, 120 minutes, 150 minutes and 180 minutes take further 1 mL samples, measure OD, dilute and plate
6. The Following day, check all plates to determine which plate at each time point is the most crowded plate on which all teh colonies can still be counted individually.
   1. The cells on these plates should be counted, and the number of colonies mutiplied by the dilution factor to calculate the number of cells in 1mL
7. Plot a calibration curve of OD against cells per mL

PCR

This protocol is desgined for use with the stratagene PfuUltra II Fusion DNA polymerase and is based in part on the PfuUltra II Fusion DNA polymerase usage manual. PfuUltra II Fusion Manual

Aim

To produce clones of two genes from B.subtilis that are too big to have synthesised by GeneArt; for use as an integration site and gene knockout (EpsE) or for their original purpose as a transcriptional regulator (XylR). Also to produce clones of sequences from vectors; for use as integration sites (AmyE), antibiotic resistance (Spectinomycin) and as a transcriptional repressor (LacI).

Equipment

Heated lid PCR machine

Thin walled PCR tube

Reagents

Reagent	Volume
Distilled H2O	13.5μL
DMSO	2.5μL
10Χ PfuUltra™II reaction buffer	5μL
dNTP mix (2.5mM each dNTP)	0.5μL
B.subtilis genomic DNA (100ng/μL)	1μL
Forward Primer (1μM)	1μL
Reverse Primer (1μM)	1μL
PfuUltra® II fusion HS DNA polymerase	0.5μL
Total Reaction Volume	25μL

Note. Template DNA should be diluted to 100ng/μL. If template DNA concentration is below 100ng/μL, 100ng of DNA should be added and the volume of H₂O to be added should adjusted to maintain a reaction volume of 25μL

If a vector is used as the template 5ng of plasmid DNA should be used instead and the volume of H₂Owater to be added adjusted

The forward and reverse primers should contain the Biobrick prefix (forward primer) and the complementary sequence to the Biobrick suffix (reverse primer) 5' of the beginning of the annealing sequence

Protocol

Add all the reagents in order (down the list) sequentially to the PCR tube mxing after each addition. Place PCR tubes into th ePCR machine and set the programme to the following set-up:

Initial Denaturation:	1 minutes at 95°C (longer for genomic DNA)
10 Cycles of:	20 second denaturation at 95°C
	20 second annealing time at Primer Tm - 10°C
	15 second extending time at 72°C (30 seconds for genomic templates)
20 - 30 Cycles of:	20 second denaturation at 95°C
	20 second annealing time at Primer Tm - 5°C
	15 second extending time at 72°C (30 seconds for genomic templates)
Final Extension:	5 minutes at 72°C

The resulting solution can then be purified using a PCR purification column or by gel electrophoresis followed by spin purification.

Preparation of XL1-Blue Electrocompetent cells

Aims

Prepaeration of E.coli cells for cloning of Biobricks and construct construction

Equipment

Centrifuge(s)

Sterile Centrifugation bottles

50mL Tubes

Large Flasks

Eppendorf Tubes

P200 Pipette

Stripettes

Reagents

1 Litre of LB medium (and appropriate antibiotics)

1-2 Litres of autoclaved and chilled ddH₂O

10% glycerol in ddH₂O, autoclaved and chilled

Dry ice bath

Protocol

Keep Everything Cold where possible

Set aside an afternoon for this, starting the culture in the morning

Check the culture while growing frequently

1. Grow up a culture of E.coli XL1-blue cells overnight
2. Add 20mL of overnight culture to 1 Litre of LB medium (containing appropriate antibiotic)
3. Grow cells while mixing at at least 225rpm until the culture reaches an OD_600nm of 0.5-0.6 (1.6-1.9×10⁸cells/mL)
   1. Test OD immediately after innoculating the Litre flask.
   2. First doubling may take 1 hour but doublings after that should be very 20-30 mins, so check often!
4. When OD is 0.5-0.6, transfer the culture to 2 sterile 500mL centrifugation bottles and cool on ice for a few minutes
5. Pellet cells in a centrifuge at 4000×g for 15 mins
6. Quickly but carefully pour off the supernatant then carefully resuspend the cells in 10mL of ice cold ddH₂O
7. Fill both tubes to about 350mL with ice cold ddH₂O
   1. Make sure the pellet is fully resuspended!
8. Repellet the cells (as before) and again discard the supernatant
9. Resuspend cells again in 10mL of ddH₂O, then fill both tubes up to about 150mL with ice cold ddH₂O
10. Repellet the cells (as before)
    1. While repelleting, fill the dry ice bath and set up eppendorf tubes (approximately 50) in a rack in the dry ice bath
11. Pour off the supernatant and resuspend the cells in 20mL of 10% glycerol (resuspend one pellet then transfer the solution to the other bottle and resuspend the second pellet)
12. Transfer the cells and glycerol solution to a sterile 50mL centrifuge tube and pellet for 15 mins at 4000×g
13. Pour off supernatant and resuspend pellet in 2mL of 10% glycerol
14. Pipette 50μL aliquots into the eppendorfs in the dry ice bath
    1. Feeze on dry ice
    2. Depending on pipetting accuracy, between 50 and 60 aliquots should be made
    3. using a repeating pipetter makes this proces much faster and reduces risk of contamination
15. Store at -70°C

Biomaterial Secretion Testing by gel

Aims

To test for expression of biomaterials in B.subtilis and to determine if the biomaterials when expressed are being secreted or are remaining within the cell. This can also be extended to find optimal conditions for production and secretion.

Equipment

Vertical Gel electrophoresis tank and equipment

Desktop centrifuge

Pipettes

Eppendorfs

Reagents

Acrylamide

Gel buffers

APS

TEMED

Water saturated butanol

ddH₂O

Laemmli buffer (4×) (SDS, 100%glycerol, Tris pH6.8, bromophenol blue, ß-mercaptoethanol (electrophoresis grade))

Lysis buffer (Tris pH 8, SDS and ddH₂O)

Coomassie Blue dye

Destain soltuion (50% methanol, 40% water, 10% glacial acetic acid)

Protein Marker

LB medium (and antibiotics)

Protocol

Buffers

Laemmli buffer

Lysis buffer

Production of Samples

• A 1mL sample of B.subtilis lacking the construct should be prepared by the following method and aliquots stored in PAGE buffer

1. Grow a culture of B.subtilis in 5mL of LB medium with antibiotics overnight
2. Take 50μL of overnight culture and inoculate 5mL of LB medium (with antibiotics) and grow until OD₆₀₀ reaches 0.4-0.6
3. When OD reaches 0.4-0.6, take a 1mL sample then induce expression using a set concentration of inducer
   1. Inducer is probably IPTG, xylose or light and each should have a specific concentration for use (determined previously)
   2. Grow at 37°C for a pre-set amount of time (probably 1 hour or 4 hours but may be more)
4. When the culture has been allowed to grow for the set amount of time (or has reached a time point if using time points), harvest 1 mL of the culture and pipette into an eppendorf.

Preparation of Samples

1. Centrifuge the sample for 5 minutes at 13000rpm
2. Being careful not to disturb the pellet, pipette off the supernatant into a separate eppendorf
3. Resuspend the pellet in μL of Lysis Buffer
4. Pipette μL of the cell lysate and the supernatant into separate tubes.
5. Mix each sample with μL of PAGE buffer ready for loading

Loading and running the Gel

1. Boil all samples at 95°C for 5 minutes
2. Carefully mix each sample before loading by rubbing tube between fingers and load 15μL of sample into the wells, taking care to note which sample is in which well on which gel (if 2 are being loaded simultaneously).
   1. When loading to gels, load along in one direction, alternating between the two gels (if both used), but beginning with the rear gel.
   2. Be careful to load sample into a single well and not contaminate adjacent wells and avoid bubbles where possible
3. Gel should be run for approximately 70 minutes at 130V, or until the blue gel front has been eluted from the bottom of the gel
   1. Check for bubble rising to ensure the gel is running correctly

Gel removal and Staining

1. When the running buffer has been eluted from the gel or has reached the very bottom, turn off the current and machine
2. Remove the lid of the PAGE tank and remove the gel holder from inside
3. Release both gels from the holder and lay them down separately in their glass cases
4. Carfeully remove the glass cover and place well away from other glass objects
5. Prepare a tray with a protein dye soltuion
6. Slowly peel the gel off the glass plate and into the protein dye solution (using the adhesive ability of the water helps)
7. Leave to stain for 20 minutes or overnight (if staining overnight, place a film or lid over the tray to reduce evaporation)
8. To destain, the stain should be poured back into the stain bottle and the gel immersed in destain solution until the destain solution is bright blue
   1. At this point, pour off the old destain and add some fresh
9. When the gel background is relatively colourless, the gel should be removed from destain and kept in water before imaging