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Contents 1. Restriction Digestion 2. Ligation 3. Transformation 4. Colony Picking 5. Glycerol Stock Preparation 6. Miniprep 7. LB Media 8. MYE Media 9. Using the TECAN Plate Reader

Protocols

Restriction Digestion

Materials

• Part A (Purified DNA to be digested, > 16ng/ul)
• Part B (Purified DNA to be digested, > 16ng/ul)
• dH2O (to fill up to 16ul total volume)
• NEB Buffer 2 (5ul)
• BSA (1ul) Restriction
• Enzymes: EcoRI, SpeI, XbaI, PstI, DpnI
• Linearized plasmid backbone (pSB1C3, 25ng/ul)

Method

1. Label two 0.6ml thin-walled tubes A and B.
2. Add 250ng of DNA samples A and B to be digested into their corresponding tube and adjust with dH2O for a total volume of 16ul.
3. Add 2.5ul of NEBuffer 2 0.and 5ul of BSA to each tube,
4. In the Part A tube: Add 0.5ul of EcoRI, and 0.5ul of SpeI.
5. In the Part B tube: Add 0.5ul of XbaI, and 0.5ul of PstI.
6. In the pSB1C3 tube: Add 0.5ul of EcoRI, 0.5ul of PstI, and 0.5ul of Dpn1.
7. Incubate the restriction digest at 37oC for 30min, and then 80oC for 20min to heat kill the enzymes.

Ligation

Materials:

• Part A (EcoRI-HF SpeI) digested DNA fragment, ( < 3ul)
• Part B (XbaI PstI )digested DNA fragment ( < 3ul)
• Digested pSB1C3 plasmid backbone (25ng/ul)
• dH2O (to fill up to 10ul total volume)
• T4 DNA ligase buffer
• T4 DNA ligase

Method:

1. Add 2ul of digested pSB1C3 plasmid backbone from the digestion procedure (25 ng)
2. Add equimolar amount of digested Part A (< 3 ul)
3. Add equimolar amount of digested Part B (< 3 ul)
4. Add 1 ul T4 DNA ligase buffer.
5. Add 0.5 ul T4 DNA ligase
6. Add water to fill up to a volume of 10 ul
7. Ligate at 16oC/30oC for 30min, and then 80oC for 20min to heat kill the enzymes.

Transformation of Competent E. coli

Materials:

• Resuspended DNA (Resuspend well in 10ul dH20, pipette up and down several times, let sit for a few minutes)
• Competent cells (DH5α and TOP10)
• Ice
• 2ml tubes
• SOC media
• 42ºC water bath
• Petri dishes with LB agar and appropriate antibiotic (Either ampicillin or Chloramphenicol)
• Plastic spreader
• 37ºC shaking and standard incubators
• 10pg/ul RFP Control (pSB1A3 w/ BBa_J04450)

Method:

1. Add 50 µL of thawed competent cells pre-chilled 2ml tubes for each part and another 50µL into a 2ml tube labelled as a control.
2. Add 1 - 2 µL of the resuspended DNA to each 2ml tube and pipet it up and down gently.
3. Add 1 µL of the RFP Control (pSB1A3 w/ BBa_J04450) to the control transformation.
4. Incubate cells on ice for 25 minutes and then perform heat shock on the cells by immersion in a 42ºC pre-heated water bath for 45 seconds.
5. Once removed, incubate the cells on ice for 5 minutes.
6. Add 200 μl of SOC media to each transformation and incubate the cells at 37ºC for 2 hours in a shaking incubator.
7. Label two petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid backbone, and antibiotic resistance. Plate 20 µl and 200 µl of the transformation onto the dishes, and spread.
8. For the control, label two petri dishes with LB agar (AMP). Plate 20 µl and 200 µl of the transformation onto the dishes, and spread.
9. Incubate the plates at 37ºC for 12-14 hours overnightin a standard incubator.

Colony Picking

Materials:

• Liquid lysogeny broth Agar
• Transformed plated cells, incubated at 37°C overnight
• Relevant Antibiotic (AMP or CAM)
• Shaking incubator at 37°C

Method:

1. To a 5ml Liquid lysogeny broth Agar culture add 5 ul of the relevant antibiotic to form the inoculum solution.
2. Pick a single, freshly grown colony with a pipette tip and inoculate the solution.
3. Cultivate the inoculum for 16 hours in the shaking incubator at 37°C.

Glycerol Stock Preparation

Materials:

• Sterile 80% glycerol solution
• Inoculum containing colonies incubated for 16 hours in in the shaking incubator at 37°C
• Vortexer
• Centrifuge
• -80°C Freezer

Method:

1. 0.5ml of this culture inoculated into sterile vial.
2. Add 0.5ml of 80% glycerol to the tube and vortex to mix.
3. Centrifuge at 8000 rpm (6800 x g) for 20 seconds.
4. Store glycerol stock in freezer at -80°C for future use.

Miniprep

Materials:

• Eppendorf tubes
• Thermo Scientific GeneJET Plasmid Miniprep Kit:
• Resuspension Solution
• Lysis Solution
• Neutralization Solution
• Wash Solution
• Elution Buffer (Not used)
• Thermo Scientific GeneJET Spin Column
• dH2O (In place of elution buffer)
• Microcentrifuge
• Vortexer

Method:

1. Place the transformed bacterial culture into an Eppendorf tube and harvest the transformed bacterial culture by centrifugation at 8000 rpm (6800 x g) in a Microcentrifuge for 2 minutes at room temperature.
2. Decant the supernatant and remove all remaining medium.
3. Resuspend the pelleted cells with 250 ul Resuspension solution then vortex the solution.
4. Add 250 ul of Lysis solution and invert the tube 4-6 times.
5. Add 350 ul of Neutralization solution to inhibit the Lysis solution and invert the tube 4-6 times. Centrifuge for 5 minutes at 10,000-14,000g (≥ 12,000 x g).
6. Transfer the supernatant to the Thermo Scientific GeneJET Spin Column and centrifuge for 1 minute at 10,000-14,000g (≥ 12,000 x g).
7. Wash the column by adding 500ul of Wash Solution and centrifuge for 30-60 seconds. This was repeated twice.
8. Transfer the column to a new Eppendorf tube and add 50ul of dH2O to elute the DNA. Incubate this for 2 minutes before centrifuging for 2 minutes at 10,000-14,000g (≥ 12,000 x g).
9. Collect the purified DNA in the flow-through.

LB Media

Used to grow overnight cultures of E. coli.

The recipe for 1l LB media is as follows:

Tryptone	15g
Yeast Extract	10g
NaCl	5g

Once these components have been added ddH2O should be used to bring the solution to 1l, followed by autoclaving.

MYE Media

MYE is a modified minimal media used to grow bacteria in the 96-well plates. MYE was used here as LB has a natural fluorescence that would interfere with our readings. It was created by Howard et al. (2013) and was modified from Schirmer et al. (2010).

The recipe for 1l MYE media is as follows:

Na2HPO4	6g
KH2PO4	3g
NaCl	0.5g
NH4Cl	2g
Tris.HCL (pH 7.25) (1M Solution)	200ml
Yeast Extract	0.5g

Autoclave the solution, then added a volume of (previously sterilised) stock solution:

CaCl2	11mg/l	100ul
MgSO4.7H2O	0.25g/l	1000ul
FeCl3.6H2O	27mg/l	100ul
ZnCl.4H2O	2mg/l	100ul
Na2MoO4.2H2O	2mg/l	100ul
CuSO4.5H2O	1.9mg/l	100ul
H3BO3	0.5mg/l	100ul
Thiamine	1mg/l	100ul
Triton X100	0.1%	1000ul

Finally add a carbon source (filter sterilized or autoclaved):

Glucose

3%

60ml

Using the TECAN Plate Reader

The TECAN Infinite 200 Pro is a multimode plate reader with a range of detection modes and functions. It was primarily used when study E. coli and the effects of our constructs in vivo. We created a method (using the accompanying software) that allowed us to scan the fluorescence and optical density of our samples. The machine also had temperature control, allowing us to keep our samples at 37 °C when they were being scanned, as well as some degree of shaking control.

By using a 96-well plate reader we were able to massively increase the range of our experiments. Ordinarily we would’ve had to have used larger cultures, and scanned them individually in a spectrometer to find their fluorescence/OD. Because we would’ve had to use larger cultures we would’ve had to use a larger volume of TNT/NG to achieve the same concentration; as TNT/NG was already in limited supply currently this would’ve massively limited the amount of experiments we were able to do.

In an example experiment: “Investigating E. coli resistance to TNT”. In this experiment we want to roughly find the level of TNT at which growth is inhibited.

Using the plate reader...

We have to use cultures of around 200 ul.

Using 12 wells, each with 100ul of TNT per ml of Media.

On the plate reader this experiment would require 240 ul TNT.

Without using the plate reader...

We have to use cultures of around 2 ml.

Using 12 falcon tubes, each with 100ul of TNT per ml of Media.

Using standard techniques this experiment would require 2.4 ml TNT.

The protocol for using the TECAN machine was initially as follows:

1. Create a 96-well plate using the desired combination of cells, media and chemicals.
2. Place the plate into the machine and run the program containing your method. This will then scan the plate and create the initial data set.
3. Remove the plate from the machine and place the plate into a shaking incubator, (800 rpm, 37 °C) until the next time point (usually 1 hour).
4. At the appointed time return the plate to the machine and scan again, creating further data sets. Repeat this until you have the data required.

Using this method we obtained our initial results. However, the flaw in this method is that during times at which we had no access to the lab (between 17:30 and 8:00 each day) we could not measure the plates, only leave them to grow in the incubator, leading to large gaps in our data in those experiments. To combat this we created a new method.

1. Create a 96-well plate using the desired combination of cells, media and chemicals.
2. Place the plate into the machine and run the program containing your new method. This method contains instructions to use the shaking/heating function of the machine to replicate the function of a shaking incubator.

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