Team:Ionis Paris/Notebook/12 10 2016

October 12th 2016

Miniprep: on DH5⍺ transformed with BBX3, BBX5, BBG3, pSB1A2-C45

Objectives

Purification and quantification of BBX3, BBX5, pSB1A2-C45 and BBG3 plasmids extracted from bacterial mini-cultures in order to construct new biobricks and to sequence them.

Materials

Mini-cultures of bacteria transformed with BBX3 (1, 2, 3), BBX5 (1, 2, 3), pSB1A2-C45 (4, 5, 6, 7) and BBG3 (1, 2, 3) realized the 11/10 (put a colony with satisfying PCR results in 5 mL LB+Cm / Amp (for pSB1A2-C45) into a 50 mL Falcon tube). From those mini-cultures, take 300 µL to realize a glycerol stock of tranformed bacteria. The 4.7 mL remaining will serve for the miniprep.

Protocol

The miniprep were realized using the QIAprep® Spin Miniprep Kit (Qiagen, ref: 27104)

Miniprep:

1. Divide each 4.5 mL bacterial O/N mini-cultures into 4 Eppendorf tubes and centrifuge all those tubes at 9,000 rpm for 3 min at room temperature. Discard the supernatant.

2. Resuspend the pellet in 62.5 μL Buffer P1 and pool the 4 Eppendorf tubes into a unique tube.

3. Add 250 μL Buffer P2 and mix by inverting the tube 6 times. The solution turns blue.

4. Add 350 μL Buffer N3 and mix by inverting the tube 6 times. The solution turns colorless.

5. Centrifuge for 10 min at 13,000 rpm.

6. Load 800 μL supernatant from step 5 to the QIAprep 2.0 spin column. Centrifuge for 1 min and discard the flow-through.

7. Add 500 µL Buffer PB. Centrifuge for 1 min at 13,000 rpm and discard the flow-through.

8. Add 750 µL Buffer PE. Centrifuge for 1 min at 13,000 rpm and discard the flow-through.

9. Centrifuge once more for 1 min at 13,000 rpm.

10. Place the QIAprep 2.0 spin column in a clean 1.5 mL microcentrifuge tube.

11. Add 50 μL Buffer EB to the center of the QIAprep 2.0 spin column, let stand for 1 min, and centrifuge for 1 min at 13,000 rpm.

12. Calculate the quantity of DNA with the Nanodrop.

13. Store the purified DNA at -20°C.

Bacteria storage :

1. Add 100 µL of glycerol to 100 µL of transformed bacteria in a clean 1.5 mL Eppendorf tube.

9 tubes of BBX3 (3 per mini-cultures)

12 tubes of BBX5 (3 per mini-cultures)

9 tubes of BBX5 (3 per mini-cultures)

9 tubes of BBX5 (3 per mini-cultures)

2. Store at -80°C.

Digestion: BBX5 and BB-B0015, pSB1A2-C45 and pSB1C3, pSB1A2-C45 and BBA

Objectives

Double digestion of pSB1A2-C45 and pSB1C3-RFP by EcoRI and PstI to create BBC45.
Double digestion of pSB1A2-C45 by XbaI and Pst1 and BBA by SpeI and PstI to create BBC3.
Double digestion of BBX5 by SpeI and PstI and BB-B0015 by XbaI and PstI to create BBX1.

NB: BBX5 is the ligation product of BB12 + X4, pSB1A2-C45 is the ligation product of pSB1A2-C5 + C4.

Materials

Stock concentrations:

pSB1A2-C45: ~150 ng/µL (from miniprep 12/10/16)

pSB1C3-RFP 3 : 104.14 ng/µL (from miniprep 19/07/16)

BBA-1: ~250 ng/µL (from mini prep 22/09/16)

BBX5: ~150 ng/µL (from miniprep 12/10/16)

BB-B0015: ~150 ng/µL (from mini prep 08/10/16 )

Quantity of DNA required for the subsequent ligations:

pSB1C3: Digestion of 100 ng (25 ng needed per ratio —> 50 ng of digested pSB1C3 needed —> 75 ng of pSB1C3-RFP needed)

pSB1A2-C45 : Digestion of 450 ng (ratio 2:1 = 29.52 ng of digested C45 needed, ratio 3:1 = 44 ng of digested C45 needed—> 74 ng of digested C45 needed —> 191 ng pSB1A2-C45 needed)

BBA : Digestion of 250 ng (25 ng needed)

pSB1A2-C45 : Digestion of 300 ng (ratio 3:1 = 25 ng of digested C45 needed —> 64 ng of BBC45 needed)

BBX5 : Digestion of 150 ng (25 ng needed per ratio —> 50 ng digested X5 needed)

BB-B0015 : Digestion of 200 ng (ratio 3:1 = 3.4 ng of digested BB-B0015 needed, ratio 5:1 = 5.8 ng of digested BB-B0015 needed) —> 9.2 ng needed of digested BB-B0015.

Protocol

Digestion:

1. In a 1.5 mL Eppendorf tube, adding in the respected order (bigger volume first and enzyme last) :



NB: The digestion were done in 20 µL.

2. Short Spin Centrifugation

3. Incubation 1h at 37°C

4. Store at 4°C before gel electrophoresis and purification

Electrophoresis for digested pSB1C3:

1% Agarose gel:

1. Put 1 g of agarose low melting point + 100 mL of TAE 1X in a bottle of 500 mL.

2. Mix and heat it 2min 30s in the microwaves. Wait the cooling of the bottle until it is tepid.

3. Add 3 µL of Gel Red 10,000X (0.3X final).

4. Flow the gel and place the combs.

5. Wait until it is solidified. Remove slowly the combs.

Drop-off:

1. Short Speed centrifugation of samples.

2. Addition of 2 µL of Purple loading dye 6X in the 20 µL of sample.

3. Drop-off 10 µL of Purple ladder and 24 µL of sample.

Plan



4. Run at 90 V.

Gel purification for digested pSB1C3:

QIAquick Gel purification kit (Qiagen, 28704), according to the protocol given by the supplier (available here)

1. Excise the DNA fragment from the agarose gel. Gel slice Weigh = 304 g

2. Add 3 volumes Buffer QG to 1 volume of gel.

3. Incubate at 50°C for 10 min until the gel slice has completely dissolved. Vortex the tube every 2–3 min to help dissolve gel. The color of the mixture is yellow.

4. Add 1 gel volume isopropanol to the sample and mix.

5. Load 800 µL of each samples to the QIAquick column. Centrifuge for 1 min at 13,000 rpm and discard flow-through. Load the rest and spin again.

6. Add 500 µL Buffer QG. Centrifuge for 1 min at 13,000 rpm and discard flow-through.

7. Add 750 µL Buffer PE. Centrifuge for 1 min at 13,000 rpm and discard flow-through.

8. Centrifuge once more for 1 min at 13,000 rpm.

9. Place QIAquick column into a clean 1.5 mL microcentrifuge tube.

10. Add 50 µL Buffer EB to the center of the QIAquick membrane, let stand for 1 min, and centrifuge for 1 min at 13,000 rpm.

11. Store the purified DNA at 4°C before the ligation.

PCR purification for digested P1, P2 and P3:

QIAquick PCR purification kit (qiagen, 28106), according to the protocol given by the supplier (available here)

1. Add 5 volumes Buffer PB (100 µL) to 1 volume of the sample (20 µL) and mix. The color of the mixture is yellow.

2. Load the sample to the QIAquick column. Centrifuge for 1 min at 13,000 rpm and discard flow-through.

3. Add 750 µL Buffer PE. Centrifuge for 1 min at 13,000 rpm and discard flow-through.

4. Centrifuge once more for 1 min at 13,000 rpm.

5. Place each QIAquick column in a clean 1.5 mL microcentrifuge tube.

6. Add 30 µL Buffer EB to the center of the QIAquick membrane, let stand for 1 min, and centrifuge for 1 min at 13,000 rpm.

7. Calculate the quantity of DNA with the Nanodrop.

8. Store the purified DNA at 4°C before the ligation.

Results

Electrophoresis:

Expected results / Obtained results:

Interpretation

The digestions were efficient, we get 2 strips at the end of the electrophoresis.

Ligation: C45 into pSB1C3, C45 into BBA, B0015 into BBX5

Objectives

Ligation of BBX5 and B0015 to obtain X1, of C45 into pSB1C3 to obtain BBC45, of C45 into BBA to obtain BBC3.
Different ligation ratios are going to be tested : 1:1, 1:2, 1:3. The molar ratios for the ligations were calculated using NEB BioCalculator (available here)

Materials

Concentrations of the different components after digestion and PCR purification :

pSB1C3: 4.3 ng/µL (129 ng / 30 µL)

C45 (EP): 3.75 ng/µL (112.5 ng / 30 µL)

BBA: 8.3 ng/µL (250 ng / 30 µL)

C45 (XP): 1.25 ng/µL (37.5 ng / 30 µL)

X5: 5 ng/µL (150 ng / 30 µL)

B0015: 3.75 ng/µL (112.5 ng / 30 µL)

1. In the following order, add :



2. Mix by pipetting

3. Incubate for 1h at room temperature

Transformation: competent DH5⍺ cells with ligation product BBC45, BBC3 and BBX1

Objectives

The objective is to transforme competent DH5⍺ cells with the ligation products of BBC45, BBC3 and BBX1.

Materials

3 aliquot of 100 µL DH5⍺ competent cells (from the 20/09/16)

Plasmid DNA : Ligation product BBC45, BBC3 and BBX1

Petri dish LB+Cm: Cm concentration = 25 µg/mL

Protocol

Experimental conditions achieved:

We need 8 LB+Cm plates + 5 LB plates

Transformation protocol

1. Thaw 2 tubes of DH5⍺ competent cells on ice for 10 min. Mix gently and carefully pipette 50 µL of cells into the 4 transformation tubes on ice.

   Add the 20 µL plasmid DNA to the cell mixture.

2. Carefully flick the tubes 4-5 times to mix cells and DNA. Do not vortex.

3. Place the mixture on ice for 30 min. Do not mix.

4. Heat shock at exactly 42°C for 45 s. Do not mix.

5. Place on ice for 5 min. Do not mix.

6. Pipette 250 µL of room temperature SOC into the mixture.

7. Place at 37°C for 60 min at 250 rpm.

8. Warm selection plates to 25°C.

9. Mix the cells thoroughly by flicking the tube and inverting.

10. Spread the corresponding volume onto each plate.

11. Incubate all the plates O/N at 37°C.

Results (obtained on the 13/10)

Expected results:

Some colonies on the petri dishes LB+Cm plated with 50 µL of bacteria transformed with the different ligation products and more on the petri dishes LB+Cm plated with 200 µL of bacteria.
A bacterial lawn on the LB petri dishes without antibiotic.
No colonies on the LB+Cm petri dish plated with bacteria transformed with no plasmid (- control).

Obtained results:

We obtained expected results.

Interpretation

The transformation worked. Colonies contain a plasmid with the Chloramphenicol resistance gene, present in pSB1C3. A PCR colonie is necessary to check the size of the plasmid present in colonies, and therefore in order to know if bacteria incorpore the correct plasmid.

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