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Team:XMU-China/Notebook Protocol
From 2014.igem.org
Protocols
Cell Culture
Antibiotic
|
Antibiotic |
Stock conc(mg/ml) |
Final conc(ug/ml) |
Solvent |
|
Chloramphenicol |
50 |
50 |
Absolute ethyl alcohol |
|
Ampicillin |
50 |
100 |
ddH2O |
|
Kanamycin |
50 |
50 |
ddH2O |
|
Tetracycline |
50 |
50 |
Absolute ethyl alcohol |
All the antibiotics above require filtration to sterilizate.
Stock in -20°C
LB Medium
|
|
Mass |
M/L |
|
Yeast Extrac |
5g/L |
0.5% |
|
Tryptone |
10g/L |
1% |
|
NaCl |
10g/L |
1% |
LB Solid Medium (on the basis of the liquid media)
|
Agar |
15g/L |
1.5% |
LB Semi-Solid Medium (on the basis of the liquid media)
|
Agar |
15g/L |
0.25% |
M63 Medium
Competent Cell
TaKaRa Competent Cell Preparation Kit
1. Prification
Activate the E.coli strain on LB-plate from glycerol stock under the condition of 37°C for 12 hours.
Pre-culture single-colony in 5 mL LB medium under the condition of 37°C, 200 rpm for 12 hours.
2. Culture
Add 200 μL pre-culture into 20 mL LB medium under the condition of 37°C, 200 rpm for 2 hours.
Place on ice for 30 min.
3. Preparation
Aliquot into sterile 1.5 mL microtube and spin down at 1500×g for 5 min at 4°C, then discard supernatant.
Gently re-suspend each pellet with 100 μL ice cold Solution A. Centrifuge at 1500×g for 5 min and discard supernatant.
Re-suspend each pellet with 100 μL ice cold Solution B.
Transformation
Add 1 μL plasmid or 10 μL ligation system into 100 μL fresh competent cells and mix gently. Leave on ice for 30 minutes.
Heat shock tubes in 42°C water bath for 90 seconds, then place them on ice for 10 minutes immediately.
Add 400 μL fresh LB medium into each tube.
Incubate for 1~2 hourswith 200 rpm shaking at 37°C. When the antibiotic of vector is ampicillin, the time of incubate is 1 hr.
Spread 100 μL on an LB solid medium plate containing the appropriate antibiotics and incubate overnight at 37°C
Confirmation
Plasmid Extraction
Omega Plasmid Mini Kit
Pellet bacterial cells from a 1.5-5 ml overnight culture.
Resuspend cells in 250 μL Solution I/RNase A.
Add 250 μL of Solution II. Mix gently by inverting 4-6 times to obtain cleared lysate.
Add 350 μL of Solution III and mix well to form white precipitate.
Centrifuge at maximum speed of at least 10000 x g for 10 min.
Transfer cleared lysate into a HiBind Mini Column placed in a 2 mL collection tube. Centrifuge for 1 min at maxi speed. Discard liquid.
Wash column with 500 μL Buffer HB. Centrifuge 1 min at maximal speed (≥ 13000 x g ). Discard liquid.
Using the same collection tube, wash column with 700μL DNA Wash Buffer diluted with ethanol. Centrifuge 1 min at maximal speed (≥ 13000 x g ).
Wash HiBind Mini Column a second time with an additional 700 μL of DNA Wash Buffer.
Centrifuge empty HiBind Mini Column for 2 min at at maximal speed (≥ 13000 x g ) to dry.
Elute plasmid DNA with 30 μL sterile water.
Digestion for Confirmation
Reaction condition: 37°C for 2 hours.
|
Single Digestion |
|
Double Digestion |
||
|
Plasmid |
5 μL |
Plasmid |
5 μL |
|
|
EcoR I |
1 μL |
Xba I |
0.5 μL |
|
|
|
|
Pst I |
0.5 μL |
|
|
10×H buffer |
1 μL |
10×M buffer |
1 μL |
|
|
ddH2O |
3 μL |
ddH2O |
3 μL |
|
|
Total Volume |
10 μL |
Total Volume |
10 μL |
|
DNA Agarose Gels (take 20mL as example)
1. Dilution
Dilute stock of 50×TAE to 1×TAE with ddH2O.
Measure 20 ml of 1×TAE buffer.
Transfer 1×TAE buffer to Erlenmeyer flask.
2. Melt
Weigh out 0.2g agarose to make 1% gel.
Transfer agarose to Erlenmeyer flask.
Melt agarose in microwave, stirring 105 seconds until completely melted.
3. Cooling
Allow gel to cool until Erlenmeyer flask can be handled comfortably.
Add 1:10 volume ratio (2μL) GelRed Nucleic acid dye to the gel and shake the Erlenmeyer flask to dye the gel well.
Pour agarose into gel tray, assemble gel pouring apparatus by inserting gate into slots.
Ligation
Digestion for Ligation
|
Insert(A) + Vector(B) |
|
Vector (B) + Insert (A) |
|||||||
|
Insert(A) |
30μL |
Vector(B) |
30 μL |
Insert(A) |
30 μL |
Vector(B) |
30 μL |
||
|
EcoR I |
3 μL |
EcoR I |
4.5 μL |
Xba I |
3 μL |
Spe I |
4.5 μL |
||
|
Spe I |
3 μL |
Xba I |
4.5 μL |
Pst I |
3 μL |
Pst I |
4.5 μL |
||
|
10×H buffer |
4 μL |
10×M buffer |
4 μL |
10×M buffer |
4 μL |
10×H buffer |
4 μL |
||
|
ddH2O |
0 μL |
ddH2O |
7 μL |
ddH2O |
0 μL |
ddH2O |
7 μL |
||
|
Total Volume |
40 μL |
Total Volume |
50 μL |
Total Volume |
40 μL |
Total Volume |
50μL |
||
Gel Extraction of DNA
Omega Gel Extraction kit
1. Melt
Perform agarose gel electrophoresis to fractionate DNA fragments.
When adequate separation of bands has occurred, carefully excise the DNA fragment of interest.
Weigh it in a clean 1.5 ml microcentrifuge tube.
Add an equal volume of Binding Buffer (XP2).
Incubate the mixture at 60°C for 7 min or until the gel has completely melted.
2. Centrifugation
Place a HiBind DNA Mini Column in a provided 2 ml collection tube. Add the DNA/agarose solution to the HiBind DNA Mini Column.
Centrifuge at 10000 x g for 1 min at room temperature. Discard the flow-through liquid.
Add 300 μL of Binding Buffer (XP2).And centrifuge at 13000 x g for 1 minute at room temperature. Then discard the flow-through liquid.
Add 700 μL of SPW Wash Buffer (diluted with absolute ethanol). And centrifuge at 13000 x g for 1 min at room temperature. Then discard the flow-through liquid
Repeat the previous step.
Centrifuge the empty HiBind DNA Mini Column for 2 min at maximal speed (≥ 13000 x g ) to dry the column matrix.
Place the HiBind DNA Mini Column into a clean 1.5 ml microcentrifuge tube. add 30 μl Elution Buffer directly onto the column matrix and incubate at room temperature for 2 minutes. Centrifuge for 1 min at maximum speed (≥ 13,000 x g) to elute DNA.
Ligation
Check the concentration of DNA fragments and vector which are going to be ligated.
Calculate the amount of insert and vector added, based on the fragment length.
Add DNA/buffer and ligase together in the EP tube.
|
Insert |
V1 μl |
|
Vector |
V2 μl |
|
10×ligation buffer |
1 μl |
|
T4 Ligase |
1 μl |
|
Add ddH2O until the total volume is 10.0μL |
|
Note that a ligation using a molar ratio of 1:3 vector to inserts.
Mix the reaction by pipetting up and down gently and microfuge briefly.
Incubate at 16°C for 5~12 hours.
PCR
PCR (Polymerase Chain Reaction)
General PCR
PCR System
|
Template |
1 μl |
|
Forward Primer |
2 μl |
|
Reverse Primer |
2 μL |
|
dNTP Mixture |
2 μL |
|
10×PCR Buffer |
5 μL |
|
r Taq (TakaRa) |
1 μL |
|
ddH2O |
37 μL |
|
Total Volume |
50 μL |
PCR Condition
|
1 |
94°C |
5min |
|
|
2 |
94°C |
30s |
30 loops |
|
3 |
Tm°C |
30s |
|
|
4 |
72°C |
1 min/kb |
|
|
5 |
72°C |
10min |
|
|
6 |
15°C |
5min |
|
Conic curve formation
Ⅰ M63 semi-solid medium preparation
|
M63 semi-solid medium/100mL |
|
|
Reagent |
Quantities |
|
KH2PO4 |
1.36g |
|
KOH |
0.42g |
|
(NH4)2SO4 |
0.2g |
|
MgSO4 |
0.012g |
|
Agar( gel strength>750 g/cm2) |
0.25g |
|
H2O |
96mL |
|
Glycerol |
0.2mL |
|
FeSO4 *1 |
10.84*10-4g |
|
D-glucose |
0.4g |
|
Asp *2 |
6.6*10-3mg |
|
Met, Leu, His, Thr (0.015g/mL) |
1mL |
Note：
1. FeSO4 *1 :Add 10μl 0.1g/mL FeSO4 to the medium
2. Asp *2 : Add 10μL 66mg/mL Asp to the medium
3. Add 96mL H2O after 0.25g Agar poured into the conical flask, shaking up, and then continue adding Glycerol using pipette.
4. Asp *2, Met, Leu, His, Thr could be promixed for convenience, and the amino acid should be added by injector through 0.22μm filter membrane, pay attention that this process must carry out after the M63 semi-solid medium is high temperature sterilized.
Ⅱ Mix-doting bacteria, inductor and inhibitor on the M63 semisolid medium plate
1. Pour 20ml M63 semisolid medium on the plate, laying up in the super clean bench for 1h30min to dry it up;
2. Doting 15μL inductor (IPTG) or inhibitor (L-Arabinose) on to plate using pipette, take care when insert the tip into the medium, then lay up in the super clean bench for 20mins to dry it up;
3. Dot 3μL Bacteria on the inductor (IPTG) or inhibitor (L-Arabinose), laying up in the super clean bench for 5mins to dry it up;
4. Have the plate sealed.
Ⅲ lineation of inductor and inhibitor on the M63 semisolid medium plate
1. Pour 20ml M63 semisolid medium on the plate, laying up in the super clean bench for 1h30min to dry it up;
2. Inhale 15μL inductor (IPTG) or inhibitor (L-Arabinose), doting along the line pre-pianted carefully, bear in mind don’t destroy the semisolid medium when moving along the line.
3. After doting, lay up in the super clean bench for 15mins to dry it up, then go on the next step.
Characterization the activity of promoter and efficiency of RBS by chemotaxis
Activation of bacteria
Transfer 50μL bacterium solution (pLac-RBS (1.0)-CheZ-TT, pLac-RBS (0.01)-CheZ-TT, pLac-RBS (0.3)-CheZ-TT) into 5ml new LB liquid medium whose chloromycetin concentration is 50ug/ml to culture for 12 hours in 37℃horizontal rotators at 200rpm.
Then transfer another 50μL bacterium solution from the LB above to another new 5ml LB liquid medium whose chloromycetin concentration is 50ug/ml to culture in 37℃horizontal rotators at 200rpm for 3 hours.
Culture
We draw three dots on a plate before, then stab 3ul bacterium medium into the M63 semisolid medium at the dots. After that culture the bacteria in constant temperature and humidity incubator at 37℃.
Measurement
Use a ruler to measure the diameter of colony from the bottom of the semi-solid medium. The initial colony radius is recorded as R1, and the radius are measured at 12h, 24h, 30h, 36h, 42h... as R2, R3, R4, R5, R6, R7…and record time and diameter. Finally processing data with excel.
Black hole
Plasmid Construction
All DNA parts applied in these experiments came from the MIT Registry of Standard Biological Parts. The new BioBrick part, PompC-cheZ (BBa_K1412010) was formed by ligating the pre-existing parts, PompC (BBa_R0082) and cheZ (BBa_K1412007). The Promega Wizard Plus SV Minipreps (Cat. #A1460) kit was used according to the manufacturer’s instructions to extract plasmid DNA. BioBrick parts were digested with restriction enzymes, EcoRI, XbaI, SpeI, and PstI to produce ‘sticky ends’ that were used to combine BioBrick parts. Digested DNA was purified using 1% agarose gel electrophoresis followed by gel purification with the QIAGEN QIAquick Gel Extraction Kit (Cat. # 28706). BioBrick parts with matching ‘sticky ends’ were ligated using T4 ligase produced by Promega. Plasmid DNA was transformed into
DH5α. Then we began a new circulation consisting of extracting plasmids, digestion and DNA gel electrophoresis to make sure the success of the ligation. Finally, the construct was sequenced for verification.
Construction of E. coli strain CL-1
The new BioBrick part, PompC-cheZ (BBa_K1412010) on pSB1C3 was introduced into E. coli strain CL-1 whose gene of cheZ had been knocked out.
E. coli strain CL-1 was offered by
I. Gradient Plate Experiments (semi-solid medium, Cm 50μg/ml)
Medium: Medium A1 (Improved)
|
Ingredients |
Quantities |
|
Yeast Extract |
0.26% |
|
Peptone |
0.27% |
|
K2HPO4 |
0.37% |
|
KH2PO4 |
0.13% |
|
NaCl |
0.05% |
|
Glycerol |
0.2% |
Sucrose concentration gradient: 0~20%, the concentration gradient was 2%.
Mid-log-phase cell suspensions preparation:
1. Overnight culture of the required strain grow at 37 °C in Medium A (Improved) containing appropriated chloramphenicol.
2. Dilute the overnight culture at the ratio of 1:100 in fresh culture (Medium A), then incubate the culture at 37 °C, until its OD590 reaches 0.2.
3. Keep it in 4 °C.
The method of preparing gradient semi-solid medium (0.25% agar)
1. Prepare 100% sucrose solution.
2. Prepare the medium with tripled ingredients amount.
3. Put the 100% sucrose solution, the prepared medium and plenty of deionized water into the autoclave for 121 degrees centigrade, 20 minutes.
4. After sterilization, put the 100% sucrose solution, the prepared medium and the deionized water into a drying oven for around 10 minutes.
5. Take 5ml tripled Medium A (Improved) into a 25ml erlenmeyer flask, then add appropriate volume of 100% sucrose solution into the erlenmeyer flask, while the shortage will be complemented by deionized water to make the total volume to be 15ml.
6. Add 15μL chloramphenicol into the erlenmeyer flask.
7. Pour the mixture into a Petri dish (85 mm dia.) as quickly as possible.
8. Leave the plate air-dried for 90 min.
9. After solidification of the media, cell suspensions from mid-log-phase cultures (3μL) were inoculated in the center of the plate.
10. Leave the plate dried in air for 15 min.
11. Leave the plate incubated at 37 °C. During the period of time, measure the diameters of the colony and taking them down.
II. Plates Crossed Experiments (semi-solid medium, Cm 50μg/ml)
Medium: half Medium A (Improved)
|
Ingredients |
Quantities |
|
Yeast Extract |
0.13% |
|
Peptone |
0.135% |
|
K2HPO4 |
0.195% |
|
KH2PO4 |
0.065% |
|
NaCl |
0.025% |
|
Glycerol |
0.1% |
The concentration of sucrose: 0%, 10%
The method of preparing liquid medium containing different concentrations of sucrose:
1. Prepare the medium with corresponding ingredients amount in the table above.
2. Add corresponding amount of solid sucrose into the liquid medium.
3. Wait for sterilization.
Mid-log-phase cell suspensions preparation:
1. Overnight culture of the required strain grow at 37 °C in Medium A (Improved) containing appropriated chloramphenicol.
2. Dilute the overnight culture at the ratio of 1:100 in fresh culture (half Medium A), then incubate the culture at 37 °C, until its OD590 reaches 0.2.
3. Keep it in 4 °C.
The method of preparing semi-solid medium with a cross (0.25% agar)
1. Prepare the medium with corresponding ingredients amount in the table above.
2. Put the medium containing agar, the liquid medium containing sucrose and plenty of deionized water into the autoclave for 121 °C, 20 minutes.
3. After sterilization, put the mediums and the deionized water into a drying oven for around 10 minutes.
4. Take 15ml of the medium containing agar into a 25ml erlenmeyer flask.
5. Add 15μL chloramphenicol into the erlenmeyer flask.
6. Pour the mixture into a Petri dish (85 mm dia.) as quickly as possible.
7. Leave the plate air-dried for 90 min.
8. After solidification of the media, draw a cross composed of two straight lines on the plate, through Intensive RBI (1μl/mm). One straight line was dotted with water, the other was dotted with liquid medium containing 10% sucrose. The length of the lines were 6 centimeters.
9. Leave the plate dried in air for 15 min.
10. Cell suspensions from mid-log-phase cultures (3μL) were inoculated at the cross point.
11. Leave the plate dried in air for 15 min.
12. Leave the plate incubated at 37 °C. During the period of time, take pictures of the colony.
Osicllation timer
To minimize the effects of pH, for bulk culture,we used Luria–Bertani (LB) medium containing 2.5 g Yeast extract, 5 g Bacto Tryptone,5 g NaCl per liter, buffered by 0.1 M HEPES (pH 8.0). To prepare semi-solid agar, 10-ml of the above medium containing 0.25% agar (Difco, Bacto agar) was poured into a Petridish, and allowed to harden at room temperature for 90 min. When rich defined medium was utilized, 0.1 M HEPES (pH 8.0) was also added and 0.5% (w/v) glycerol wasused as the carbon source. Unless otherwise stated, all other reagents were from Sigma.
In order to make chemotaxis effect more obvious, we spot bacteria in the half thickness in the centern of the palte and cultivated. All experiments were carried out at 37 °C.
References
