Plasmid purification

Goal

The aim of the plasmid purification step is to recover the plasmid produced by the bacteria (cloning strain).
The different step are use to throw other bacterial components off (proteins, cell wall, genomic DNA, etc).

Preparation

1. Cell culture
Cultivate cells in LB medium overnight.

2. Cell harvesting
Set saturated E.coli LM culture into 2 mL tubes. Centrifuge at 11 000 x g for 30 secondes. Discard as much as supernatant as possible.

3. Cell lysis
Add 250 μL of Buffer A1 (resuspension buffer). Resuspend the cells with a vortex or a pipette.
Add 250 μL of Buffer A2 (lysis buffer). Mix gently by inverting the tube 6 - 8 times. Incubate at room temperature until lysate appears clear.
Add 300 μL of Buffer A3 (neutralisation buffer). Mix thoroughly by inverting the tube 6 - 8 times .

4. Lysate clarification
Centrifuge at 11 000 x g for 5 minutes . Repeat this step until supernatant is not clear.

5. DNA Binding
Place a NucleoSpin Plasmid Column in a Collection Tube of 2 mL and set the supernatant from the last step. Centrifuge at 11 000 x g for 1 minute. Discard flow-through and place the column back into the collection tube.

6. Membrane washing
Add 600 μL of Buffer A4 (wash buffer) previously supplemented with ethanol. Centrifuge ar 11 000 x g for 1 minute. Discard flow-through and place the column back into an empty collection tube.

7. Dry membrane
Centrifuge at 11 000 x g for 2 minutes and discard the collection tube.

8. DNA Elution
Place the column in a 1,5 mL and add 50 μL de Buffer AE (elution buffer). Incubate at room temperature and centrifuge at 11 000 x g for 1 minute.

Test

Before the storage of the tubes at -20°C, measure the concentration with nanodrop.

Figure 1: Figure 1: Nanodrop test after a plasmid purification

If the concentration is between 30 and 50 ng/μL, concentrate your DNA with SpeedVac concentrator or any other method.
If the concentration is below 30 ng/μL or if 260/280 ratio or 260/230 ratio is not correct, make another purification.

260/280 ratio and 260/230 indicate the purity of DNA (or RNA).
Nucleic acids absorb at 260 nm while proteins and phenols absorb at 280 nm and carbohydrates at 230 and other contaminents at 230 nm.
For DNA, a 260/280 ratio around 1,8 is concider to be pure and ange 260/230 ratio must be between 2,0 and 2,2.

Gel electrophoresis analysis

Principle

Electrophoresis is a method using electrical field to separate DNA or RNA sequence by size. Smaller the fragment is, the more it migrates on the gel.
Using a DNA ladder, we can know the size of DNA sequence and then check if we have the sequence we wanted.

Preparation

Put 0.5 g of Agar in 50 mL of TAE 1X and heat until the mix is melted.
Once the agar have dissolved and the mixture have cooled down, put 0.3 mL of EtBr in it.
Pour the mixture on the mould, put the comb and wait until the gel polymerized.
Put the gel in the electrophoresis unit (with TAE 1X).

Put your DNA sample (with loading dye) in the PCR well, do not forget the kB ladder. After the migration (~30 to 45 minutes depanding to your fragment size), observe the gel with UV light.

PCR Purification

Goal

The aim of the PCR purification step is to recover DNA from PCR and other enzymatic reaction mixtures.

Preparation

1. Mixture preparation
Add 1:1 volume of Binding Buffer to completed PCR mixture (e.g. if you have 100 µL of reaction mixture, add 100 µL of Binding Buffer). Mix thoroughly and check the solution's color: a yellow color color indicates an optimal pH for DNA binding.
If the color of the solution is orange or violet, add 10 µL of 3 M sodium acetate, pH 5,2 and mix. The color will become yellow.
If the DNA fragment is superior to 500 bp, add a 1:2 volume of 100% isopropanol (e.g. if you have 100 µL of PCR mixture combined with 100 µL (total amount of 200 µL), add 100 µL isopropanol). Mix thoroughly.

2. Binding DNA
Transfer up 800 µL of the solution from step previous step to the GeneJET purification column.
Centrifuge for 30-60 seconds, then discard the flow-through.

3. Membrane washing
Add 700 µL of Wash Buffer (previously dilted with ethanol) to the purification column.
Centrifuge for 30-60 seconds, then discard the flow-through and place the purification column back into the collection tube?

4. Dry membrane
Centrifuge the empty GeneJET purification for 1 minute to completely remove any residual wash buffer

5. DNA Elution
Transfer the GeneJET purification column to a clean 1,5 mL tube.
Add 50 µL of Elution Buffer to the center of the GeneJET purification column membrane and centrifuge for 1 minute. Discard the GeneJET purification column, test with the Nanodrop and store the purified DNA at -20°C.

BioBrick Assembly

Goal

Once you have your constructions on your plasmids, you need to biobrick them before sending them to the registry.

Preparation

Restriction

For restriction you need: distilled water, Bovine Serum Albumin (BSA, used to stabilize restriction enzymes), Restriction Buffer 1,2,3 or 4 (buffer 2 works for all combinations of EcoRI-HF, XbaI, PstI, SpeI), two restriction enzymes per sample.
Determine which restrictions enzymes and Buffer you need using openwetware.org¹.
Determine the DNA-concentration of the vector and the insert.
Prepare restriction mix for all restrictions,for each reaction add:

• 12,5 μl od distilled water
• 5,0 μl of Buffer 1,2,3 or 4
• 0,5 μl of BSA
• 1,0 μl of restriction restriction enzyme 1
• 1,0 μl of restriction restriction enzyme 2

Then, for each reaction, add:

• 20 μl restriction mix previously prepared to a PCR-tube
• 1 μg of DNA.
• Distilled water, until your reach a total volume of 50 μl

PCR Purification

Purify the restriction digest, and then determine the concentration of the purified DNA with Nanodrop.
For more information, see the PCR purification protocol.

Ligation

Prepare ligation mix.
Prepare two extra reactions, “No insert” and “No ligase” that will be used as negative controls.

For each reaction,add:

• 50 ng of vector-DNA.
• Insert in three times molar excess, then add 3·50·(length of insert/length of vector) ng of insert-DNA.
• Distilled water, until your reach a total volume of 20 μl

Transformation Test

• If no colonies form in both controle, the restriction was perfect for both the restriction enzymes.
  
• If colonies form in both cases, neither of the restriction enzymes worked well. Most of the transformants will contain the vector only.
  
• If the “No insert” control resulted in many colonies but “No ligase” did not, one of the enzymes worked well and the other did not. Most of colonies will carry the self-ligated vector.
  

Re-isolate colonies.
Check that colonies have been successfully transformed (e.g. by PCR).
Inoculate those colonies in overnight culture for further use.
Send your part to iGEM registry !

BioBricks Characterization overview

TECAN Analysis

Goal

The Tecan is a plate reader that is able to characterize our bacteria of interest by measuring, for example, growth rate (OD) and/or protein production (GFP). The software saves the measurements over time, thus allowing to study the kinetics of the studied phenomena. With a 96 wells plate (8x12), we are able to test various conditions.

Steps

The hardest part when you use a plate reader to characterize your parts is to carefully choose your controls and anticipate the timings of your bacterial pre-cultures.

We tested various controls during this summer:

• TOP10 growth control
  
• BL21 growth controlFor GFP expression: GFP expression under the control of a pLac promoteLac operator in a delta-LacI strain and GFP expression under the control of a Fur operator in a Δ-Fur strain.

Preparation

Medium preparation

LB medium emit a side signal. As turbidity (OD) and fluorescence of our sample are measured, M9 medium is use instead.

Composition for 50 mL of:

Once the mixture is prepared, the medium must be filtered to be sterilised using 0.22 µm filter.

Pre-culture preparation

In a 15 mL tube, add 2 mL of M9 medium and inoculate BL21 cells (expression strain) from glycerol
To inhibit the expression of sfGPF, use M9 with iron, instead of classical M9.
After one night of culture, refresh the precultures by diluting them 200 times in M9 medium (with iron and carbenicillin). After 8 hours of culture, prepare your wells plate according to the following scheme:

Technical triplicate are made to see the variation induced by the manipulator.
Biological duplicate are made to determine the natural variation of the process observed.

Microfluidic experiment

Goal

The aim of microfluidic analysis is to trap bacteria in a chip, with a continuous medium. Hence, the behaviour (e.g. GFP production) of bacteria in exponnential phase can be analyse with microscope during long acquisition (>24h).

Chip preparation

Figure 1: Microfluidic ship mould
(designed by Boris Kirov)

Mix gently 36 g of Polydiméthylsiloxane (PDMS,condensed formula: (C2H6OSi)n ) with 3.6 g of curring agent (which make the PDMS polymerize).
Put in a 50 mL tube and centifugate it at 13 000 for 1 minute to remove bubbles from the mixture.
Pour the mixture on the chip mould and put it under a vacuum pump.
Once there is no more bubbles that go out of the mixture, put it in the oven at 80°C for 2 hours.
Separate the different chips and make the holes of the input and outpout of channels with a punch.
Remove all the dust on chips surfaces. Then put chips and glass slides on plasma oven.
After that, put quickly the chip on glass slide and put them in the oven at 80°C for 2 hours.

Bacteria preparation

Make 30 mL of preculture of your strain of interest in LB medium with the recquired antibiotics to avoid contamination.
Let it growth overnight.
Centrifugate and resuspend the pellet in 3 mL of LB medium with the recquired antibiotics and mild detergent
Inject bacteria in the chip with needle and syringe.

Figure 2: Microfluidic system acquisition from microfluidic ship (A: two ship), microscop (B) and injecting system (C)

Figure 3: A Escherichia coli (TOP 10 strain) in a microfluidic trap.
At the top and bottom of the picture, the flow of medium go throught the 2 channels.

Acquisition

Wash the chip with your recquired medium (in our case: M9 with or without iron).

Homologous recombination

Goal

Preparation

Strain Preparation

Transform the PTKred Plasmid¹ with the strain of interest.
Start an over night culture with the strain transformed with PTKred at 30°C.
Dilute the overnight culture in a ratio of 1 mL/100 mL and add IPTG (isopropyl β-D-1-thiogalactopyranoside; final concentration of 2 mM) and Spectinomycin (60 mg/mL).
Grow up to OD600: 0.5 then make electrocompetent cells .

Integration

Prepare the PCR fragment to be integrated flanked by 50 bp genomic sequences.
Gel-purify the product and transform with the electrocompetent PTKred strain.
Recover in LB for 2 hours with IPTG.
After 2 hours incubation add the selective drug which should be inserted in the genom (for example kanamycin if you have kan R cassette inserted) and incubate overnight in liquid culture at 30°C.
Next day, culture should be very turbid or the recombination did not work. Plate streak 50 μL of the overnight culture on a plate containing selection drug which in this case is kanamycin and grow overnight at 30°C.
Next day pick a colony and make glycerol stocks

Elimination of PTKred plasmid

Inoculate the strain that you have made by Homologous Recombination in LB overnight with spectinomycin at 30°C.
Adding spectinomycin in this step ensures that the plasmid will not integrate in the genome in a rare occurrence.
Dilute 1/100 and grow at 42°C for 4 hours and then plate sreak 50 μL of the culture on LB plate at 42°C.
Next day, pick colonies and grid plate on LB then spectinomycin and grow at 37°C.

Flip Recombinase and Anti-biotic Resistance Gene

Transform a cured strain with PCP20 plasmid
Grow overnight in LB with Cam at 30°C overnight.
Dilute 1/100 and grow for 3 hours at 37°C.
Streak plate on LB plates and grow over night at 42°C.
Repeat the grid.

Siderophore detection

Goal

Once our bacteria is transformed with the plasmid with the Fur Binding Site and Lac I and the two plasmids with Enterobactins genes, we need to check if our system really work. The production of siderophores can be detected visualy with Blue Agar Chrome Azurol S (CAS). Without siderophore in the medium, CAS and Hexadecyltrimethylammonium bromide (HDTMA) complexes with ferric iron, producing a blue color. When a bacteria strain produce siderophore, the medium color change from blue to orange.

Preparation

Blue Dye

Solution 2
Dissolve 0,0027 g of FeCl₃-6H₂0 in 10 mL of 10 mM HCl.

Solution 3
Dissolve 0,073 g of HDTMA in 40 mL of distilled water.

Mix
Mix solution 1 with 9 mL of solution 2. Then mix with solution 3.
Solution should have a blue color.
Autoclave and store in a bottle.

Mixture solution

Minimal Media 9 (MM9) Salt Solution Stock
Dissolve 15 g of KH₂PO₄, 25 g of NaCl and 50 g of NH₄Cl in 500 mL of distilled water.

NaOH Stock
Dissolve 25 g of NaOH in 150 mL of distilled water.
pH should be around 12.

20% Glucose Stock
Dissolve 20 g of glucose in 100 mL of distilled water.

Casamino Acid Solution
Dissolve 3 g of Casamino acid in 27 mL of distilled water.
Extract with 3% 8-hydroxyquinoline in chloroform to remove iron.
Filter with a 0,22 µm millipore.

CAS agar preparation

Add 100 mL of MM9 salt solution to 750 mL of distilled water.
Bring pH up to 6 and dissolve 32,24 g of piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES); PIPES will not dissolve between pH of 5.
Add 15 g of Bacto Agar.
Autoclave and the cool to 50°C.
Add 30 mL of sterile Casamino acid solution and 10 mL of sterile 20% glucose solution to MM9/PIPES mixture.
Slowly add 100 mL of Blue Dye solution along the glass wall while mixing thoroughly.

Bacteria

Figure 1: CAS plate with four different bacteria plated. Moving clockwise from top left: Micrococcus luteus, Staphylococcus aureus, Enterobacter aerogenes, Escherichia coli