Team-iGEM SJTU BioX 201

Protocols-iGEM SJTU BioX 201

Content

Polymerase chain reaction
DNA Gel Electrophoresis
Restriction Digestion
Ligation
Plate preparation
Liquid culture media preparation
Competent cell preparation for transformation
Transformation
Competent cell preparation for electroporation
Electroporation
Colony PCR
Quantitative PCR
Induction of protein expression
Fluorescent measurement
Experiments conducted with kits
Equipment

1.PCR

Materials:

(1)10×Plus Neo Buffer
(2) MgSO4 solution
(3) dNTPs
(4) Template
(5) F/R-primer
(6) Plus neo DNA polymerase

Procedures:

(1) Combination of all

Total volume	20μL
10×buffer	2μL
dNTPs	2μL
MgSO4	1.2μL
Forward primer	0.6μL
Reverse primer	0.6μL
enzyme	0.4μL
template	1μL
H2O	12.2μL

(2) Set the PCR machine to run the following steps:

Total volume	temperature(℃)	time	cycle
initial denaturation	98	2min	1
denaturation	98	15s	35
annealing	Tm	30s
extension	68	30s for 1000bp
final extension	68	7min	1
incubate	4	infinite	1

2.DNA Gel Electrophoresis

Materials:

(1) Agarose
(2)TBE Buffer
(3)Gel board
(4)4S Green Plus Nucleic Acid Stain
(5)DNA 10×Loading Buffer
(6)Electrophoresis tank
(7) Sample
(8) Marker 2000/5000/15000

Procedures:

(1) Prepare an agarose gel with an appropriate concentration for the fragment (0.8%-1.5% (w/v)) in TBE buffer.
(2) Heat the mix until completely dissolved.
(3) Add 10000×nuclear acid stain to cooled mix.
(4) Pour the mix into gel board and insert the comb.
(5) Remove the comb after the gel solidifies and transfer to the electrophoresis tank.
(6) Add samples into pores with 10×DNA Loading Buffer.
(7) Run the gel for 20 min at adequate voltage.

3. Restriction Digestion

Materials:

(1) Restriction enzyme: Thermo Fisher FastDigest Restriction Enzyme
(2) 10×FD Green Buffer
(3) DNA samples
(4) Sterile water

Procedures:

Note: the components listed below are for double digests.

Component	volume or mass
(1) 10×FD Green Buffer	2μL
(2) DNA sample	600 ng for plasmid or up to 200 ng for PCR product
(3) Restriction enzyme	1 μL each
(4) Sterile water	add to 20μL
(5) Set up the reaction following the above table and incubate at 37℃ for 15-30 min and do the gel electrophoresis or purification later.

4.Ligation

Materials:

(1) T4 DNA Ligase
(2) 5×T4 DNA Ligase Buffer
(3) Vector plasmid
(4) Insert DNA fragment
(5) Sterile water

Procedures:

Component	volume or mass
(1) 5×T4 DNA Ligase Buffer	4μL
(2) Vector plasmid	50ng
(3) Insert DNA	require molar ratio of 3:1 to 7:1 (insert: vector)
(4) T4 DNA Ligase	0.4μL
(5) Sterile Water	add to 20μL
(6) Make the reaction system and incubate at room temperature for 4-6 hours.

Note: we highly recommend NEBbioCalculator calculation of vector and insert DNA volume.

5.Plate preparation

Materials:

(1) Tryptone
(2) Yeast extract
(3) NaCl
(4) Agar
(5) Sterile water
(6) Desired antibiotic

Procedures:

Component	volume or mass
(1) Tryptone	10g
(2) Yeast extract	5g
(3) NaCl	10g
(4) Agar	15g
(5) ddH2O	add to 1L
(6) Sterilize at 121℃ for 20 min.
(7) Desired antibiotic: suggested concentration

Antibiotic	suggested concentration(ng/μL)
Streptomycin	50
Chloramphenicol	25
Ampicillin	50
Kanamycin monosulfate	50
Tetracycline hydrochloride	100
Apramycin sulfate	50

6.Liquid culture media preparation

Materials:

(1) Tryptone
(2) Yeast extract
(3) NaCl
(4) Sterile Water

Procedures:

Component	volume or mass
(1) Tryptone	10g
(2) Yeast extract	5g
(3) NaCl	10g
(4) ddH2O	add to 1L
Sterilize at 121℃ for 20 min.

7.Competent cell preparation for transformation

Materials:

(1) ice
(2) sterile 100mM CaCl2
(3) bacterial culture

Method:

(1) Overnight bacterial culture 1:100 inoculate in Erlenmeyer flask.
(2) Incubate until early exponential stage (OD600 0.2-0.3).
(3) Turn on the centrifuge and set temperature to 4℃, start pre-cooling.
(4) Take out the culture and put on ice. Put sterile CaCl2 on ice.
(5) Transfer the bacterial culture into centrifuge tube, 4000rpm centrifuge for 3min at 4℃.
(6) Discard the supernatant, add 10mL ice cold sterile CaCl2 and resuspend the cell softly, 4000rpm centrifuge for 3min at 4℃.
(7) Discard the supernatant, add 4mL ice cold sterile CaCl2 and resuspend the cell softly, 4000rpm centrifuge for 3min at 4℃.
(8) Discard the supernatant and add 100mM CaCl2 according to the quantity of bacteria (1mL competent cells out of 50mL bacterial culture) and resuspend softly.
(9) Put 50μL competent cells each into 1.5mL EP tube, store at -80℃.

8.Transformation

Materials:

(1) Competent cells
(2) LB broth
(3) Ice
(4) Selection plates with corresponding antibiotics.

Method:

(1) Put 50µL competent E. coli cells on ice for 10 minutes
(2) Add 20 µl DNA from a ligation reaction mix or 10-100 ng plasmid
(3) Incubate the mixture on ice for 30 minutes
(4) Heat shock at exactly 42°C for exactly 45 seconds（DH5α），90 seconds (BL21(DE3))
(5) Place on ice for 2 minutes
(6) Add the mixture into 500 µL LB broth
(7) Incubate at 37°C and 200-250 rpm for one hour
(8) For ligation reaction DNA: 500 µl of each transformation reaction onto a selection plate. For known plasmid: 100-200 µL of each transformation reaction onto a selection plate
(9) Incubate overnight at 37°C with plates upside down.

9.Competent cell preparation for electroporation

Object: To prepare competent cell for electroporation

Instrument: centrifuge(type), clean bench

Materials:

(1) ice
(2) sterile ddH2O
(3) sterile 10% glycerol
(4) bacterial culture

Method:

(1) Over night bacterial culture 1:100 inoculate in a Erlenmeyer flask.
(2) Incubate until early exponential stage (OD600 is 0.2-0.3).
(3) Turn on the centrifuge and set temperature to 4℃, start pre-cooling.
(4) Take out the culture and put it on ice. Put sterile water and sterile 10% glycerol on ice.
(5) Transfer the bacterial culture into centrifuge tube, 4000rpm centrifuge for 5min at 4℃.
(6) Discard the supernatant, add 10mL ice cold sterile water and resuspend the cell softly, 4000rpm centrifuge for 5min at 4℃.
(7) Discard the supernatant, add 10mL ice cold sterile 10% glycerol and resuspend the cell softly, 4000rpm centrifuge for 5min at 4℃.
(8) Discard the supernatant, add 5mL ice cold sterile 10% glycerol and resuspend the cell softly, 4000rpm centrifuge for 5min at 4℃.
(9) Repeat step (8).
(10) Discard the supernatant and add 10% glycerol according to the quantity of bacteria (1mL competent cells out of 50mL bacterial culture) and resuspend softly.
(11) Put 50μL competent cells into each 1.5mL EP tube, store at -80℃.

10. Electroporation

Material: 50μL competent cell, 1μL linear DNA or circular plasmid, 1mL ice-cold LB

Instrument: clean bench, electroporator

Method:

(1) Thaw competent cells on ice.
(2) Chill electroporation cuvettes on ice.
(3) Turn on the electroporator and set the parameters.
(4) Add 1-2μL of linear DNA (with a concentration of 200ng/μL or above) or plasmid (with a concentration of 50ng/μL or above) into 50μL competent cells and mix gently with the end of a pipette tip.
(5) Transfer the cell/DNA mixture into a chilled cuvette, wipe off moisture and water from outside of cuvette using a kimiwipe.
(6) Insert the cuvette into the electroporator, and press the pulse button.
(7) Immediately add 1mL ice-cold SOB medium (or LB medium) to the cuvette after the end of the pulse.
(8) Transfer it into a 1.5mL EP tube.
(9) Shake the tube at optical condition for 60 minutes.
(10) Centrifuge at 2500rpm for 5min.
(11) Remove 950μL LB and resuspend the else, spread onto selective plate (LB plate with corresponding antibiotics).
(12) Incubate overnight at optimum condition.

11. Colony PCR

Materials:

(1) 2×Colony mix
(2) Primer
(3) Plate with colony
(4) Enhancer
(5) ddH2O

Method:

(1) Combination of all reagents:

Total volume	20μL
2× Colony mix	10μL
Forward primer	0.5μL
Reverse primer	0.5μL
template	-(colony)
enhancer	2μL
H2O	7μL

(2) Set the PCR machine to run the following steps:

stage	temperature(℃)	time	cycle
initial denaturation	95	3min	1
denaturation	95	10s	35
annealing	Tm+5	10s
extension	72	10s for 1000bp
final extension	72	5min	1
incubation	4	infinite	1

12. Quantitative PCR

Materials:

(1) Tsingke qPCR mix
(2) Rox
(3) Template
(4) F/R primer
(5) ddH2O

Method:

(1) Combination of all reagents:

Total volume	20μL
2× qPCR mix	10μL
Forward primer	0.8μL
Reverse primer	0.8μL
template	3μL
Rox	0.4μL
H2O	5μL

(2) Briefly centrifuge
(3) Set qPCR machine using default program for SYBR green.

13. Induction of protein expression

13.1 IPTG

(1) Inoculate 1% overnight bacterial culture in Erlenmeyer flask.
(2) Incubate for 2h to reach early exponential stage (OD600 is 0.2-0.3).
(3) Add IPTG to final concentration of 0.5 mM.
(4) Incubate overnight at 20℃

13.2 Tetracycline

(1) Inoculate 1% overnight bacterial culture in Erlenmeyer flask.
(2) Incubate for 2h to reach early exponential stage (OD600 is 0.2-0.3).
(3) Add IPTG to final concentration of 100 ng/μL.
(4) Incubate overnight at 20℃

13.3 AHL induction and preparation for copy number measurement.

(1) Inoculate 1% overnight bacterial culture in Erlenmeyer flask. (2) Incubate for 2h to reach early exponential stage (OD600 is 0.2-0.3). (3) Add AHL to final concentrations of 10-9, 10-8, 10-7, 10-6 M. (4) Take 500μL of the culture every 30min. (5) Centrifuge at 4000rpm for 5min immediately after taking out. (6) Discard the supernatant and wash with 500μL PBS. (7) Heat at 100℃ for 10min. (8) Store at -20℃ for at least 20min. (9) Centrifuge at 6500rpm for 1min. (10) Remove the supernatant into a clean 1.5mL EP tube. (11) Store at -20℃.

14. Fluorescent measurement

Materials:

96-well ELISA plate
96-well luminescence plate
Microplate reader
PBS buffer

Procedures:

(1) Measure OD600 value of samples.
(2) Adjust OD600 of all samples to appropriate value.
(3) Wash with PBS to rid samples of LB for 2-3 times.
(4) Measure fluorescence with microplate reader.

15.Culture and induction in hydrogel panel

Materials:

(1) 96-well ELISA plate
(2)Microplate reader
(3)Alginate
(4)PDB
(5)CaCI2
(6)Tetracycline

Procedures:

1.Centrifuge the bacteria culture in its early exponential stage at 4000rpm for 3min.
2.Discard supernatant and resuspend it with 5mg/ml alginate at reach OD 0.15~0.2.
3.Seed 100ul alginate mixed with alginate into 96 well plate.
4.Add 40ul交联剂（0.025M CaCl2 or 1.25mg/ml PDB）to each well.
5.Add 10ul tetracycline of 2mg/ml to each well.
6.Measure OD600 and florescence under microplate reader at time intervals.

16.Kits

Kits were bought for DNA extraction from gel, Plasmid extraction, Purification of PCR product and Gibson assembling. Below are the kits we used, all these experiments were conducted according to protocols provided.

1) DNA extraction from gel: DC301 FastPure Gel DNA Extraction bought from Vazyme, protocol link http://www.vazyme.com/companyfile/83.htmL;
2) Plasmid extraction: DC201 FastPure Plasmid Mini Kit bought from Vazyme, protocol link http://www.vazyme.com/companyfile/85.htmL;
3) Purification of PCR product: the same kit as plasmid;
4) Gibson assembling: GenBuilderTM Cloning Kit bought from Genscript, protocol link https://www.genscript.com.cn/product/documents?cat_no=L00701&catalogtype=Document-PROTOCOL

17.Equipment

PCR instruments: ProFlex
Electrophoresis tank:
Shaking Table
Incubator
Clean Bench:
Eppendorf centrifuge: Thermo Fisher sorval Legend Micro 17R
Nanodrop:
Microplate reader:
Electroporator:

Team:SJTU-BioX-Shanghai/Protocols

Content

1.PCR

Materials:

Procedures:

2.DNA Gel Electrophoresis

Materials:

Procedures:

3. Restriction Digestion

Materials:

Procedures:

4.Ligation

Materials:

Procedures:

5.Plate preparation

Materials:

Procedures:

6.Liquid culture media preparation

Materials:

Procedures:

7.Competent cell preparation for transformation

Materials:

Method:

8.Transformation

Materials:

Method:

9.Competent cell preparation for electroporation

Materials:

Method:

10. Electroporation

Method:

11. Colony PCR

Materials:

Method:

12. Quantitative PCR

Materials:

Method:

13. Induction of protein expression

13.1 IPTG

13.2 Tetracycline

13.3 AHL induction and preparation for copy number measurement.

14. Fluorescent measurement

Materials:

Procedures:

15.Culture and induction in hydrogel panel

Materials:

Procedures:

16.Kits

17.Equipment