Difference between revisions of "Team:Aix-Marseille/Experiments/Protocols"

Revision as of 16:34, 15 October 2016

1 Protocol #0 : iGEM General Protocol
2 Protocol #1 : Preparation of competent bacteria cells
3 Protocol #1bis : Preparation of Tbf1 and Tbf2 buffers
- 3.1 Preparation of 50 mL of Tbf1 Buffer
- 3.2 Preparation of 8 mL of Tbf2 Buffer
4 Protocol #2 : Transformations
- 4.1 Plasmids transformation
- 4.2 Ligation transformation
5 Protocol #2 : PCR and electrophoresis
- 5.1 PCR
- 5.2 Electrophoresis
6 Protocol #3 : Plasmid DNA purification
7 Protocol #3 : Cloning protocol for IDT sequences
8 Protocol #4 : Digestion and ligation for verification and BioBrick Assembly
9 Protocol#5 : PCR clean-up and gel extraction
10 Protocol#5 : Generalised transduction using phage P1
11 Protocol#6 : Cadaverin HPLC analysis
12 Protocol#7 : SLIC (sequence- and ligation-independent cloning)
- 12.1 Procedures
13 Protocol#8 : Swimming test
14 SLIC Oligo table

Protocols

Protocol #0 : iGEM General Protocol

Obtaining Biobricks
- EITHER on IDT →
  - Order on IDT when the sequences are not too long (chemical synthesis).
  - The received biobricks are lyophilized, they must be resuspended in water (cf protocole).
- OR by PCR →
  - When sequences are too long and costly to order on IDT, or when a sequence must be added to an existing biobrick :
    - Order primers
    - synthesize sequence by PCR (cf protocole Q5)
    - add the suffixes and prefixes during the synthesis « E, X » and « S, P » (E=EcoRI, X=XbaI, S=SpeI, P=PstI) and homologue sequences to plasmids
    - Do a SLIC (cf protocole) to insert the wanted sequence in the wanted plasmid
    - the Biobrick is obtained
Transform the Bacteria with the Biobrick
1. E/S Digestion of the Biobrick from the original plasmid and reinsertion in the E/S pre-restricted wanted plasmid.
2. Transform the pre-cultivated competent cells, via a thermal shock (DH5-α, TGI, BL21).
3. Add an LB-agar in a petri dish and cultivate the bacteria in rich medium added with antibiotics of interest.
Test the Transformation
1. Run the obtained bacteria culture DNA in a PCR and an electrophoresis gel (qualitative assessment)
2. Purify plasmids (by miniprep Kit) from the previously obtained positive colonies.
3. Do an E/P digestion verification to see if the inserted biobricks have the correct size (On only 100ng of plasmids)
Assemble two biobricks
- “Digestion protocol BioBrick Assembly Kit” et “Ligation protocol BioBrick Assembly”
1. Do an E/S digestion on the first biobrick and an X/P on the second
2. Ligate the two biobricks then insert them into the plasmid
Transforming Bacteria with a new BioBrick
1. E/P Restriction of the biobrick and ligation into a E/P restricted plasmid
2. Transform the pre-cultivated competent cells, via a thermal shock (DH5-α, TGI ou BL21).
3. Pour an LB gel in a petri dish and cultivate the bacteria in an antibiotic rich medium
Test the Transformation
1. Run the obtained bacteria culture DNA in a PCR and pour an electrophoresis gel (qualitative assessment)
2. Purify plasmids (by miniprep Kit) from the previously obtained positive colonies.
3. Do an E/P digestion verification to see if the inserted biobricks have the correct size (On only 100ng of plasmids)
4. Send to sequencing
Repeat steps 4, 5, 6 until obtaining the wanted sequence

Protocol #1 : Preparation of competent bacteria cells

Note : Everything should be done in sterile conditions and on ice (4°C). For 100ml of competent cells

Grow a culture of bacteria in LB medium until it reaches OD600 = 0.5.
- While it grows, prepare Tbf1 and Tbf2 buffers.
Centrifuge cells for 10 minutes at 3500g at 4°C
Resuspend the pellet slowly in 50mL of Tfb1 buffer.
Centrifuge 5 min at 3500g at 4°C.
Resuspend the pellet in 8 mL of Tbf2 buffer.
Incubate 15 min in ice.
Aliquot 200µL of cell suspension in sterile eppendorf tubes and conserve it at -80°C.

Protocol #1bis : Preparation of Tbf1 and Tbf2 buffers

For 100 mL of culture:

Preparation of 50 mL of Tbf1 Buffer

KAc 1M	1.5 mL
MnCl2 0.5M	5 mL
KCl 1 M	5 mL
CaCl2 0.1M	5 mL
Gly 80%	0.93 mL
H2O	32.56 mL

Preparation of 8 mL of Tbf2 Buffer

NaMOPS 0.2M	400 µL
CaCl2 0.1M	6 mL
KCl 1 M	8 mL
Gly 80%	1.5 mL
KCl 1M	80 µL
H2O	500 µL

Protocol #2 : Transformations

Plasmids transformation

Add 20 ng of plasmid to 100 μL of competent cells thawed in ice
Incubate 30-45 min in ice
Thermal shock : put tubes in the Thermomixer at 42°C for 2 min
Incubate 5 min in ice
Add 900 μL {of LB
Incubate 1 hour at 37°C with agitation
Spread 100 μL on LB limp (with antibiotic)

Ligation transformation

Add 20 ng of ligation product to 100 μL of competent cells thawed in ice
Incubate 30-45 min in ice
Thermal shock : put tubes in the Thermomixer at 42°C during 2 min
Incubate 5 min in ice
Add 900 μL of LB
Incubate 1 hour at 37°C with agitation
Centrifuge 5 min at 5000 rpm
Eliminate 850 μL of medium
Spread 150 μL on LB limp (with antibiotic)

To make negative control, follow the same procedure but without adding plasmids and spreading 300 μL

Protocol #2 : PCR and electrophoresis

PCR

	25µL reaction	50µL reaction	100µL reaction	Final concentration
EconoTaq PLUS 2X Master Mix	12.5µL	25µL	50µL	1X
Forward Primer (100 pmol/μL)	0.5µL	0.5µL	1µL	1 pmol/μL (1 μM)
Reverse Primer (100 pmol/μL)	0.5µL	0.5µL	1µL	1 pmol/μL (1 μM)
DNA Template*	0.5µL	1µL	1µL	?
Water nuclease-free	[ ] μL qsp 25μL	[ ] μL qsp 50µL	[ ] μL qsp 100μL

*One colony in 50 μL of Water. 15-20 min at 80°C. 5 min at 96°C.

Cycling steps

Cycling step	Temperature	Time	Number of cycles
Initial denaturation	98°C	3 min	1
Denaturation	95°C	10 sec	X27
Annealing	50-65°C	40 sec
Extension	72°C	1 min/kb
Final extension	72°C	1 min/kb	1

Electrophoresis

Preparation of 0.8% agarose gel

Put 0.8g of agarose powder into 100mL of TAE 1X.
Melt in microwave and sitr occasionnally.
Let cool until you can take it into hands.
Flow the support with the gel.
Wait until it's solid.

Sample preparation and migration

Put the gel in the migration tank filled with 0.5X TAE.
Mix the samples with 1X marker***.
Migrate 30 min at 100V.

Revelation

Put the gel under UV.

Protocol #3 : Plasmid DNA purification

Protocol used for a Macherey-Nagel purification kit, for more details see [http://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NS.pdf here]. For better results use gloves and if so do not use gloves.

Step	Actions
Cultivate and harvest cells	Centrifuge at 11,000g for 30 sec
Cell lysis	Add 250µL of buffer A1 Add 250µL of buffer A2 Incubate at room temperature up to 5 min 300µL buffer A3
Clarification of the lysate	Centrifuge at 11,000g for 5 min
Bind DNA	Place a purification column in a collection tube (2mL) and carefully decant supernatant from the previous step, to a maximum of 750µL Then centrifuge at 11,000g for 1 min and discard the flow-through. Put the column back in the collection tube Repeat this step to load the rest of the supernatant if need be
Wash silica membrane	Add 600µl of buffer A4 Centrifuge at 11,000g for 1 min
Dry silica membrane	Centrifuge at 11,000g for 2 min
Elute DNA	Place the columns in an 1.5mL eppendorf and add 50 µL of buffer AE and incubate at room temperature for 1 min Centrifuge at 11,000g for 1 min

To measure your DNA concentration, use a Biospec Nano apparel or Nanodrop apparel. Make the blank with 1µL of pure water and use 1µl of sample. Make sure to clear the lens with water and with a special paper in order to no line the lens.

Protocol #3 : Cloning protocol for IDT sequences

Resuspension

Resuspend gBlocks Gene fragment to a final concentration of 10 ng/µL in TE or AE (elution buffer). The tube contain 1000 ng of gBlocks Gene fragment so you have to add 100µL of AE Mix well, a vortex shall be used

Digestion E/P

1. Digest 100 ng in 50µL

component	50 µL of reaction
DNA	10 µL
Buffer 2 (10X)	5 µL
BSA (100X)	0.5 µL
H2O	33 µL
EcoRI	1 µL
PstI	1 µL

2. Incubate for 45 min at 37°C 3. Incubate 20 min at 80°C (inactivation of restriction enzyme)

Ligation

1. Ligate 50 ng vector with a 3X molar excess of gBlocks Gene fragments in fresh T4 DNA ligase buffer diluted to 1X and 400 U T4 DNA ligase for a total volume of 20 µL

component	20 µL of reaction
PSBIC3 (or other vector) at 12.5 ng/µL	2 µL
insert digested (at 37.5 ng/µL)	12 µL
T4 Buffer	2 µL
T4 ligase 400 U	1 µL
H2O	3 µL

2. Incubate 2h at room temperature

Transformation

Add 10 µL of ligation’s product to 100 µL of competent cells thawed in ice (TGI cells)
Incubate 45 min in ice
Thermal shock: put tubes in the thermomixer at 42°C during 2 min
Incubate 5 min in ice
Add 900 µL of LB
Incubate 1 hour at 37°C with agitation
Centrifuge 5 min at 5000 rpm
Eliminate 850 µL of supernatant
Suspend the pellet in the 150µL of remaining medium
Spread 150 µL on LB limp (with antibiotic)

To make negative control, follow the same procedure but without add plasmids and spread 300 µL

Protocol #4 : Digestion and ligation for verification and BioBrick Assembly

Digestion (verification) protocol

DNA	Between 50 and 100 ng
EcoRI-HF	0.2 µL
PstI	0.2 µL
10X NEBuffer 2	2 µL
H2O	QS 20 µL

Incubate all digest reactions at 37°C for 1 hour and then add 3 µL of SES 4X and migrate 30 min at 150V on a 1% agarose gel.

Digestion protocol BioBrick Assembly Kit

Upstream part :

Upstream part plasmid	500 ng
EcoRI-HF	1 µL
SpeI	1 µL
10X NEBuffer 2	5 µL
H2O	To 50 µL

Downstream part, optional, for 3A strategy

Downstream part plasmid	500 ng
XbaI	1 µL
PstI	1 µL
10X NEBuffer 2	5 µL
H2O	To 50 µL

Destination plasmid :

Destination plasmid	500 ng
EcoRI-HF	1 µL
PstI	1 µL
DpnI	1 µL
10X NEBuffer 2	5 µL
100X BSA	0.5 µL
H2O	To 50 µL

Incubate the three restriction digest reactions at 37°C for 10 minutes and then heat inactivate at 80°C for 20 minutes.

Ligation protocol BioBrick Assembly

Upstream part digestion	2 µL
Downstream part digestion	2 µL
Destination plasmid digestion	2 µL
10X T4 DNA ligase buffer	2 µL
T4 DNA ligase	1 µL
H2O	11 µL

Incubate at RT for 1 hour.

Protocol#5 : PCR clean-up and gel extraction

	PCR clean-up	Gel extraction
PCR clean-up, DNA clean-up, or single stranded DNA clean-up: Adjust binding condition Gel extraction: Excise DNA fragment / solubilize gel slice	200 μL NTI/ 100 μL PCR	200 μL NTI/ 100 mg gel (melt at 50°C for 5–10 min)
Bind DNA	Spin at 11,000g during 30s in a clean-up column
Wash silica membrane	Add 700 μL NT3 and spin at 11,000g during 30s Recommended: 2nd wash again with 700μL NT3 spin 11,000g 30s
Dry silica membrane	Spin at 11,000g 1 min
Elute DNA	15–30 μL NE RT 1 min 11,000g 1 min

Protocol#5 : Generalised transduction using phage P1

Step1 : P1 lysat on the given strain

From a overnight starter, (3mL in 2YT medium + appropriated antibiotiques) from the strand holding the transduct mutation, make a 1/100 dilution in 3mL 2YT medium and add 150µL of Cacl2 0.1M ( without antibiotique corresponding of the transduct allele). Incubate at 37°C (under agitation) until obtening a DO600=0.5-1 (more 0.5) Add 150µL of a P1 lysat wt Incubate at 37°C without agitation (water bath) during 20min Incubate at 37°C under agitation during 3 or 4h (even overnight) Divide the culture in 2 eppendorfs of 2mL (=2*1.5mL) Add 150µL chloroform then vortex Centrifuge 10min at 4°C and 6000rpm Transfer 1mL supernatant into 2 news eppendorf of 2mL Add 10µL chloroform (under a fumehood) in each eppendorf, vortex and keep at 4°C (option: to increase the titration level, you can do a second cycle)

Step2 : Transduction P1

The eve, prepare a culture of the recipient strain in 3mL 2YT medium (+eventual appropriated antibiotique) The D-day, start a culture from the recipient strain (1/50 dilution) in 3mL 2YT + 150µL of CaCl2 0.1M ( prepare the LB-citrate 5mM requiered and LBagar- appropriated AB + citrate 2mM petri dishes) Incubate until reaching a DO600 = 1 or + (2.10^6 bact/mL) Prepare 3 tubes eppendorf of 2mL (1 control with 500µL cells, 1 with 10µL P1 + 500µL cells, 1 with 100µL P1 + 500µL cells) Infect during 20min at 37°C (heat wuthout agitation) Add 1mL steril LB-citrate 5mM and heat at 37°C during 50min under agitation (700-750rpm) Centrifuge 5min at 5000rpm (RT) Remove supernatant sterilely with a P1000 and resuspend in 1mL steril LB-citrate 5mM Spread on LBagar- appropried AB + 2mM citrate Incubate ON at 37°C

Step3 : Clones isolation

Isolate clones on petri dishes (approprieted AB + citrate 2mM) Incubate at 37°C

Step 4 : PCR screen and transplantation

Test clones by PCR Transplant the good clones on LB-AB appropriate petri dishes

Protocol#6 : Cadaverin HPLC analysis

Reaction of lysine and cadaverine produced by biotransformation with whole cells

The assays were performed in a total volume of 500µL, containing 500mM sodium acetate buffer (ph = 6.0), 10mM L-lysine, 0.1 mM pyridoxal-5-phosphate, and 20µL whole cells, at 37°C in a water bath. The reaction was stopped after 24h by the addition of 10µL ethanol. The reaction mixtures were centrifuged and the 30µL of supernatant was applied to derivatization to determine the amounts of residual lysine and product, i.e cadaverine.

Derivatization reaction

Diamine deritatives were obtained by the reaction of 180µL of borate buffer 0.05M (pH9), 60µL of 100% methanol, 47µL diethyl ethoxymethylenemalonate without any pretreatment. The samples were heated at 70)C for 2h to allow complete degradation of excess DEEMM and derivatization.

HPLC analysis

After derivatization with diethyl ethoxymethylmalonate, analyses were performed on a high performance liquid chromatograph (HPLC Agilent technologies, 1260 Infinity) consisting of a binary pump, an inline degasser, an autosampler and a column thermostat. Chromatographic separation was carried out by reverse-phasechromatography on a C18 column maintained at 35°C. Mobile phase A was composed of 100% acetonitrile, and B was made up of 25mM aqueous sodium acetate buffer (pH = 4.8). The flow rate of 1mL/min was used, with the following gradient program : 0-2min, (20-25% A; 2-32min, 25-60%A; 32-40min, 60-20%A. Detection was carried out at 284nm.

Protocol#7 : SLIC (sequence- and ligation-independent cloning)

Procedures

1. Digest vector with restriction enzyme during 3h and purify the linearized vector with a commercial PCR purification kit. Elute DNA with elution buffer or 10mM TrisCl, pH 8.0-8.5. Do nnot elute the DNA with water or TE. Measure the concentration of the vector.

2. Amplify your gene of interest by PCR using primers with 15 mer (or inferior) homology extension to the linearized vector end. We usually use 15bp homology for single fragment cloning, and 20bp for multiple fragment cloning. Purify the linearized vector with a commercial PCR purification kit. Elute the DNA with water or TE. Measure the concentration of the insert(s).

3. Mix the linearized vector and insert at a molar ratio of 1.2 ina 1.5mL tube. An examble is shown as follows. (Vector to insert molar ratio of 1/1 for single fragment cloning, 1:2:2 for multiple fragments cloning. AN example of 3 fragments cloning is shown below with vector: insert 1: insert 2 molar ratio as shown is 1:2:2 as shown in Fig. 4A).

	Stock concentration	Volume added	Final concentration
Linearized vector	100ng/µL	1µL	10ng/µL
Insert 1	40ng/µL	1µL	4ng/µL
Insert 2	40ng/µL	1µL	4ng/µL
10X BSA		1µL	1X
10X NEB Buffer 2		1µL	1X
H2O		Up to 10µL

4. Add 0.2µL of T4 DNA polymerase (3U/µL, NEB) to the mixture and incubate 2min at room temperature

Protocol#8 : Swimming test

Soft geloses have been made by adding 0.3% agar in LB medium. Once dried, each bacteria strain has been spotted using a tooth pic on the petri dishes and incubated for 3h in a 37°C room.

SLIC Oligo table

DesA slic forward	cgctaaggatgatttctgGAATTCGCGGCCGCTTCTAGATGCGCTCGCACTTGCTT
desA slic reverse	ttgcccttttttgccggaCTGCAGCGGCCGCTACTAGTATTATTAGGAGGCACGGTC
desB slic forward	"a rentrer"
desB slic reverse	ttgcccttttttgccggaCTGCAGCGGCCGCTACTAGTATTATTACACTGCAAATTC
desC slic forward	"a rentrer"
desC slic reverse	ttgcccttttttgccggaCTGCAGCGGCCGCTACTAGTATTATTAGGCTGAAACCGC
desD slic forward	"a rentrer"
des D slic reverse	ttgcccttttttgccggaCTGCAGCGGCCGCTACTAGTATTATTAACGCCCGGCTAA
FliC E. coli slic forward	cgctaaggatgatttctgGAATTCGCGGCCGCTTCTAGATGGCACAAGTCATTAAT
FliC E.coli slic reverse	ttgcccttttttgccggaCTGCAGCGGCCGCTACTAGTATTATTAACCCTGGAGCAG
FliC desulfo slic forward	cgctaaggatgatttctgGAATTCGCGGCCGCTTCTAGATGTCACTGGTTATCAAT
FliC desulfo slic reverse	ttgcccttttttgccggaCTGCAGCGGCCGCTACTAGTATTATTAGCCGCCGAGAAG
CsgA E. Coli slic forward	"a rentrer"
CsgA E. Coli slic reverse	"A rentrer"

@@ Line 445: / Line 445: @@
 |-
 | Bind DNA
-| colspan="2"|Spin at 11,000g 30 s
+| colspan="2"|Spin at 11,000g during 30s in a clean-up column
 |-
 | Wash silica membrane
-| colspan="2"|700 μL NT3 11,000g 30 s Recommended: 2nd wash 700μL NT3 11,000g 30 s
+| colspan="2"|Add 700 μL NT3 and spin at 11,000g during 30s <u>Recommended</u>: 2nd wash again with 700μL NT3 spin 11,000g 30s
 |-
 | Dry silica membrane