Experiments

Plasmid Prep

1. Find the cryovial with the plasmid (in our case pBSANDlux, light pink in colour), KEEP THIS ON DRY ICE AT ALL TIMES. Using a sterile loop, scrape some of the frozen cells from the cryovial so that the loop becomes inoculated and streak this onto an LB agar plate supplemented with an appropriate antibiotic for selection (in our case, ampicillin) to obtain single colonies. Do this in a flow hood and keep the tube on dry ice.
2. Once streaked, incubate the plate at 37^oC O/N in a static incubator
3. Set up a 2 mL O/N culture of LB medium supplemented with Ampicillin, inoculate this with a colony from the streak plate with the plasmid, allow this to grow O/N at 37^oC in a shaking incubator at 200 rpm. – Allow it to grow for 12-18 hours ideally.
4. Follow the plasmid prep kit instructions on how to prep the plasmid
5. Quantify the amount of plasmid using a nanodrop (in µg/µL) WRITE THE CONCENTRATION AND DATE ON THE SIZE OF THE EPPENDORF AS WELL AS THE DATE.

Golden Gate assembly

Reagents

• 10X T4 Ligase Buffer
• 10X CutSmart Buffer
• 400,000 U/ml T4 DNA ligase
• 20,000 U/ml BsaI-HFvs2
• X ng of plasmid
• X ng of insert (this might be an amplified PCR fragment or a purchased gBlock

1. Use the NEB ligation calculator to determine the mass of plasmid you need. NEB recommends 27 fmol of plasmid to ensure optimal ligation – the mass in ng calculated form the fmol concentration will depend on the size of the plasmid. Using the online calculator, and click on the tab mass -> moles, then click moles to mass. plasmid pBSANDlux (which is 10927 bp) at 27 fmol gives a mass of 182.3 ng (this is the final concentration required).
2. To get to a final concentration 182.3 ng. Divide this value by the concentration of pBSANDlux determined by the nanodrop. E.g if you have a plasmid concentration of 100 ng/uL based on the nanodrop, then you need to add a volume of 1.96 µL of purified pBSANDlux to the GoldenGate mix below.
3. For your insert, which is the NOT gate gBlock, use the NEB ligation calculator. Into this, entire the size of the plasmid (10927 bp) the size of the insert NOT gate gBlock (814 bp) and then the calculated mass required for this plasmid (182.3 ng). This will calculate the concentration of the NOT gate gBlock that needs to be added to the GoldenGate mix. When I did this, the calculator gave me a concentration of 13.58 ng of the NOT gate gBlock (for a 1:1 ratio of insert to plasmid). Note: The gBlocks have been resuspended to a concentration of 10 ng/uL, so if you need a final concentration of 13.58 ng, you need 1.35 uL of the NOT gate gBlock in your Golden Gate mix.
4. For the GoldenGate mix, add reagents to the following amounts as required. MAKE SURE YOU KEEP ALL THE REAGENTS ON ICE. See the table below, and make up the mix in a PCR microfuge tube.

Reagent	Volume	Final Amount
Insert(s)	1.35µL	13.58ng
Plasmid	X µL	196.1 ng
10x T4 Ligase Buffer	1 µL	1x
10x CutSmart Buffer	1 µL	1x
400,000 U/mL T4 DNA ligase	0.25 µL	10,000 U/mL
20,000 U/mL BsaI-HFvs2	0.5 µL	1000 U/mL
Water	X µL	(Make up to 10 µL)

Using the example concentrations on the previous page, the volume of plasmid would be 1.96 uL and the mass in ng would be 196.1 ng. For the fragment, the volume of purified PmaeN would be 2.85 uL and the mass would be 2.85 ng.

Golden Gate Cycles

Temperature (oC)	Time (mins)
37	10	x 10 Cycles
16	10
16	30
50	5
80	10
4	Hold

After the thermocycler step, the resulting mix (10 µL) can be transformed directly into competent Escherichia coli DH5α cells using standard heat-shock and plated onto the appropriate selective medium. – If you don’t want to do this the same day as the golden gate, then keep the mixture in the -20^oC freezer.

Transformation of the assembled plasmid into E. coli DH5α

Once you have assembled the plasmid pBSANDlux with your NOT gate gBlock, you need to transform it into E. coli so the plasmid with your circuit can be replicated and lots of copies made so that we can extract out the plasmid, and put it into B. subtilis to see if it works.

1. Thaw a tube of DH5-alpha Competent E. coli cells on ice for 10 minutes.
2. Add 10 µl of the Golden Gate mix directly to the cells. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
3. Place the mixture on ice for 15 minutes. Do not mix.
4. Heat shock at exactly 42°C for exactly 90 seconds. Do not mix.
5. Place on ice for 5 minutes. Do not mix.
6. Pipette 950 µl of room temperature SOC medium into the mixture.
7. Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
8. Warm selection plates to 37°C. – These are LB agar plates supplemented with ampicillin (100 µg/mL)
9. Mix the cells thoroughly by flicking the tube and inverting, then perform three10-fold serial dilutions in SOC medium
10. Spread 100 µl of each dilution onto a selection plate and incubate overnight at 37°C. Alternatively, incubate at 30°C for 24-36 hours or 25°C for 48 hours.

Once you’ve seen that you have colonies, the colonies which should have the insert will be white, anything that is red means there was plasmid that wasn’t cut by the enzyme and the RFP cassette wasn’t removed.

Making a Patch Plate

Once you have inspected your transformation plate, you need to make a patch plate. A patch plate is just an LB agar plate with antibiotic (for the plasmid screening in E. coli, this needs to be an LB plate with ampicillin) that you use to pick colonies from your transformation plate and streak them onto a new fresh plate so the cells can grow to a bigger cell density.

1. To do this, pick a white colony (using a pipette tip) and carefully streak a small line onto the fresh LB agar plate with ampicillin which will be your patch plate. I STRONGLY recommend that on your patch plate, you draw a grid of about 10 squares which are carefully labelled numerically. Into each grid you will make a small streak of a single colony from your transformation plate. I.e pick a colony and streak it into square one, pick another colony and streak it into square two, etc.
2. Incubate the plate for a 3-4 hours at 37’C and check to see if you can see any cells beginning to grow. Now, you can do the colony PCR using the GoTaq Green MasterMix. See the reaction mixture below – If not, incubate overnight if you have a busy day.

The patch plate will be used to do a colony PCR.

Colony PCR

Colony PCR is used to screen bacterial colonies to see if have a plasmid integrated, have a plasmid which successfully had a gBlock or PCR fragment ligated into it, if a genetic deletion has been successful, amongst many other applications. (you’re all scientists, so you know all the applications of screening cells)

Reagent	Volume	Final Concentration
GoTaq(R) Green Master Mix, 2x	12.5 µL	1x
Upstream Primer, 10µM	0.25-2.5 µL	0.1-1.0 µM
Downstream Primer, 10µM	0.25-2.5 µL	0.1-1.0 µM
DNA Template	1-5 µL	<250ng
Nuclease-Free Water made up to	25 µL	NA

For plasmid pBSANDlux, the primers you need to add are IGB03/IGB04 – these are primers which anneal either side of the RFP sites in pBSANDlux so will show you if an insert has been placed in between the BsaI sites. (This will test if your Golden Gate was successful). This is better than using insert specific primers because if you assemble multiple inserts at the same, you can check for the incorporation of multiple inserts using these standard primers. – The annealing temperature for these primers is 60^oC

Setting up the Colony PCR

1. The DNA template here is the actual bacterial colony. First set up your PCR tubes (don’t forget a positive control (pBSANDlux plasmid, 10 ng) and a negative control (no plasmid), and label then with the numbers from the grid (i.e tube 1 = colony 1 from grid 1 of the patch plate, tube 2 = colony 2 from grid 2 of the patch plate). I strongly recommend you set up a mastermix for this. Multiple the volumes above by the number of tubes you will have, and add everything into a single 1.5 mL Eppendorf and then aliquot 25 uL into each PCR tube. DO NOT ADD THE pBSANDlux PLASMID INTO THE MasterMix. Make the mastermix first, aliquot 25 uL into the PCR tubes, and then to your tube labelled negative control, you add nothing else, but to your positive control, you add 1 uL of 10 ng of pBSANDlux plasmid. Dilute the stock of plasmid accordingly to achieve this.
2. To add the bacterial colony to the other tubes, use a pipette tip (attached to a pipette) and just slightly touch the colony of the patch plate so that the pipette tip gets some bacterial cells onto the tip. Then, put this tip that now has the cells from, for example colony 1, into the PCR tube and pipette up and down and move the tip of the pipette which touched the cells around the inside walls of the PCR tube in the colony PCR mix. Do this for all the tubes. Be careful not to pick a large density of cells as it means you end up with a lot of debris at the bottom of the gel you will use to visualise if you have your gBlock inserted.

PCR Conditions

The annealing temperature for the Fwd and Rev primers the check for the insertion of genes into the Golden Gate site is 60^oC.

Temperature (oC)	Time
95	10 mins
95	30 secs	x30 Cycles
60	30 secs
72	90 secs
72	1 min

1. Whilst the PCR is running, make a 1% agarose gel using 1X TAE buffer (50 mL gel) and be sure the gel has enough wells to accommodate the number of samples you want to run including your ladder, negative control and positive control.
2. Once the PCR is done, load the PCR mixtures directly into the wells of your gel and run for 20 mins at 140 V. The GoTaq has green dye in it so you don’t have to add loading dye separately.
3. The band size for the addition of the xylose inducible NOT gate is 883 nt.
4. Pick the colony which has the correct band size, and make an O/N culture of the one with the correct band size
5. Plasmid prep this plasmid and send it for sequencing using the eurofins kit.
6. Check the sequencing result against the reference plasmid (in our case, the xylose NOT gate-luciferase reporter).

Transformation of Bacillus subtilis

1. Using the enzyme ScaI, cut 1-2 µg of your transformant (in our case, the pBSANDlux-NOT gate) for 1 hour as per the NEB enzyme digestion protocol (use a 50 uL reaction) – look this up online. After 1 hour, heat the enzyme mix on a heat block to 80°C for 10 mins to inactive the enzyme.
2. Inoculate 10 ml MNGE to OD₆₀₀ = 0,1 (or simply 1/100) with overnight culture of B. subtilis W168. I usually do, 250 µL of B. subtilis cells in LB medium and 9.75 mL of MNGE medium (see below). Add this into an autoclaved conical flask, cover with foil, label with your name, the culture and date.
3. Let this grow to OD600 = 0.9-1.3 at 37°C with agitation (at least 200 rpm!). Use the 1 cm cuvette and as a blank use LB medium. Once OD600 has been reached, take 400 µL of cells. To grow to OD600 = 0.9-1.3 takes about 4 hours.
4. Once you reach the right OD, take 400 µL of your cells from the conical flask and add to a sterile 15 mL falcon tube. Make x2 version of this.
5. Add DNA (1-2 µg linearized plasmid) to one falcon tube, and to the other add nothing.
6. Let it grow for 1 h at 37’C (shaking incubator)
7. Let grow for 1 h
8. Plate on selective media – LB agar with chloramphenicol. – incubate O/N at 37’C in a static incubator.

10x MN-Medium:

• 136g K2HPO4 (x3 H2O)
• 60g KH2PO4
• 10g Na-citrat (x2 H2O)

MNGE-Medium:

• 9.2 ml 1 x MN-Medium (920 µl 10x MN + 8.28 ml sterile water)
• 1 ml Glucose (20%)
• 50 µl K-Glutamat (40%)
• 50 µl Fe[III]- ammonium-citrate (2,2 mg/ml)
• 100 µl Tryptophan (5 mg/ml)
• 30 µl MgSO4 (1M)
• (100 µl threonine (5 mg/ml) for strains carrying an insertion in thrC)

Expression Mix:

• 500 µl yeast extract (5%)
• 250 µl casamino-acids (CAA) (10%)
• 250 µl H2O
• 50 µl Tryptophan (5 mg/ml)