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1% NaCl, 1% tryptone, and 0.5% yeast extract
Configuration volume: 1000 ml
① Weigh: weigh 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl, respectively, in a beaker;
② Solubilization: 700 ml dH2O was added in a beaker and stirred with a glass rod so that the powder was completely dissolved;
③ Bring to volume: pour the solution into a measuring cylinder and add water to 1000 ml
④ Aliquot: aliquot the medium to a small tube (5 ml/tube)
Sterilization: sterilize at 121 ° C for 20 min.
mlcc-F: (gene-specific primer TM calculated as: 63.2 ° C homology arm length: 15 BP homology arm GC content: 60.0% enzyme digestion site: ggatcc)
5'- cagcaaatgggtcgcggatccATGAGCAACCCGTACCAGCG -3'
mlcc-R: (gene specific-primer TM calculated as: 60.6 ° C homology arm length: 15 BP homology arm GC content: 66.7% enzyme digestion site: ctcgag)
5'- gtggtggtggtggtgctcgagTTATTGGCAGTGGCGATTATTG -3'
mlcc-bsla-R: (gene specific primer TM calculated as: 61.0 ° C homology arm length: 15 BP homology arm GC content: 66.7% digestion site: ctcgag)
5'- gtggtggtggtggtgctcgagTTAGTTGCAACCGCAAGGCT -3'
mlcc-mhfbi-R: (gene specific primer TM calculated as: 65.3 ° C homology arm length: 15 BP homology arm GC content: 66.7% enzyme digestion sites: ctcgag)
5'- gtggtggtggtggtgctcgagTCAAGCACCGACGGCGGT -3'
mlcc-mhgfi-R: (gene specific primer TM calculated as: 60.9 ° C homology arm length: 15 BP homology arm GC content: 66.7% digestion sites: ctcgag)
5'- gtggtggtggtggtgctcgagTCAGACGTTAACCGGAACAGATC -3'
Primer processing: centrifugation was performed at 12000 rpm for 10 min, and primers were diluted to 10 × by pressing the message on the walls of primer tubes plus ddH2O μM final concentration.
The basic components of the PCR reaction include: template DNA (band amplified DNA), primers, 4 deoxyribonucleoside triphosphates (dNTPs), DNA polymerase, and appropriate buffers.
PCR consists of three basic reaction steps of denaturation----annealing----extension:
① High temperature denaturation of template DNA: after the template DNA is heated to about 94 ℃ for a certain time, the template DNA double strand is unwinded, become a single strand, so that it binds to the primer, for the next round reaction preparation;
② Low temperature annealing (renaturation) of template DNA to primers: after denaturation of template DNA into single strands by heating, the temperature is reduced to about 55 ° C, and the primers bind to the complementary sequence of the single strand of template DNA paired;
③ Isothermal extension of primers: the DNA strand complementary to the template is synthesized by extension from the 5 'end → 3' end of the primer with dNTPs in the presence of DNA polymerase (optimal activity at about 72 ° C). Each cycle underwent denaturation, annealing, and extension with a 1-fold increase in DNA content.
*PCR reaction system (50 µ L):
>Template <0.5μg
>The upstream primer (10μM） 1μl
>The downstream primer (10μM） 1μl
>ten×PFU buffer (containing Mg2 +) 5μl
>dNTP（10mM） 1μl
>PFU DNA polymerase 1.25μl
>ddH2O up to 50μl
>PCR reaction procedure (three-step):
>Pre denatured at 95 ° C for 5 min,
>30 recycles：95℃ 30 s，60℃ 45 s，72℃ 1kb/2 min
>72℃ 10 min
>Hold at 4 ℃.
Samples of post amplification DNA products were collected and spiked with 6×The DNA loading buffer was mixed for loading, electrophoresed at 120 V constant voltage for 20 ~ 30 min, and electrophoretic pictures were saved（Agarose gels were prepared by mixing: 1×TAE buffer + agarose, heat in microwave oven until agarose all dissolve then cool to below 60 ℃ add nucleic acid dye mix, 1.5g/100ml agarose, and 20μL/100mL nucleic acid dye）
The fragment of interest was recovered according to the procedure of the Agarose Gel Recovery Kit and finally eluted with 30μL ddH2O after heating and stored at - 20 ℃.
Preparation of plasmid DNA: extract the desired vector plasmid with the Column Plasmid DNA Miniprep Kit.
1) Vector double digestion and purification
① Vector digestion reaction system (50 µ L):
>ten× Green Buffer 5 µL
>BamH I 2.5 µL
>Xho I 2.5 µL
>Plasmids ~ 2.5μg
>ddH2O was made up to 50 µ L
The reaction system was inactivated by water bath at 37 ° C for 1 h and metal bath at 85 ° C for 10 min
② Purification of digested products: the digested products were recovered according to the procedure of agarose gel recovery kit and finally digested with 30μL ddH2O elution, store at - 20 ℃.
Using seamless cloning kit to connect the target gene and the plasmids after double digestion, the reaction system was as follows:
>Plasmid gene of interest (10 µ L):
>The target DNA fragment yμL
>Vector DNA xμL
>two×Basic Assembly Mix 5 μL
>ddH2O was supplemented to 10μL
10μL reaction system, the vector to individual insert addition is recommended to be between 0.01 and 0.25 pmols, and the optimal molar ratio of vector to individual insert is 1:2.
Pmols = mass ng / (fragment length BP× 0.65 kDA)
As:
100 ng of a 2000 bp fragment equals 100 / (2000×0.65) of about 0.08 pmols.
100 ng of a 5000 bp fragment equals 100 / (5000×0.65) to about 0.03 pmols.
Also do the positive control cloning reaction system:
>Control Insert 1 μL
>Linearized pUC19 Control Vector 1 μL
>two×Basic Assembly Mix 5 μL
>Nuclease-free Water 3 μL
Mix it gently, 55℃ for 10min. After the reaction is finished, immediately place the centrifuge tube on ice to cool for several seconds.
① Remove 1 tube of trans1-t1 (DH5α）Competent cells, thawed on ice;
② Per 50.0 μL competent cells were added to approximately 2μL of reconstituted product (the remaining reconstituted product was stored at 4℃, if transformation failed, reconstitution was possible every other day), mix by gently flicking the wall of the centrifuge tube (vortex is prohibited), and leave on ice for 30 min;
③ Heat shock: place the centrifuge tube in a 42℃ water bath and heat shock for 30 s, note: do not shake the centrifuge tube;
④ Ice bath: quickly transfer centrifuge tube to ice bath, place 2-3min;
⑤ Resuspend: add 450 µ l per tube of competent cells μL of sterile, antibody free LB liquid medium and incubated at 37 ° C in a shaker at 250 rpm for 1 h with shaking;
⑥ Coating: after centrifugation at 3500 rpm for 5 min, 400μ50. After resuspension of the bacteria, spread the remaining broth evenly all over the LB plate (containing antibiotics);
Culture: after the coating liquid on the plate was completely absorbed, the culture dish was inverted and the colonies were observed after placing into the 37 ℃ constant temperature incubator for 12 ~ 16h.
① Individual clones were picked to 10μL sterile water, vortex or blow mix;
② Take 1.0μl mixture at 25μl PCR system with appropriate forward and reverse primers to identify positive clones
*PCR reaction system (50 µ L):
>Template 1μl
>The upstream primer (10μM） 0.5μl
>The downstream primer (10μM） 0.5μl
>ten×Buffer (containing Mg2 +) 2.5μl
>dNTP（10mM） 0.5μl
>PFU DNA polymerase 0.6μl
>ddH2O up to 25μl
>PCR reaction procedure (three-step):
>Pre denatured at 95 ° C for 5 min,
>30 recycles：95℃ 30 s，60℃ 45 s，72℃ 1kb/2 min
>72℃ 10 min
>Hold at 4 ℃.
① Single clones were picked and inoculated into LB tubules(containing the resistance) and cultured overnight;
② The corresponding overnight tubules from each clone were removed and 600μl of broth was added to 400μl 50% glycerol, do well for labeling, and store at - 80 ℃ until cold. The remaining bacterial fluid was used for plasmid extraction using the miniprep kit for column plasmid DNA, and the plasmid was identified by double digestion followed by running on Gel.
>ten× Green Buffer 2 µL
>BamH I 1 µL
>Xho I 1 µL
>Plasmids ~ 1μg
>ddH2O was made up to 20 µ L
The reaction system was inactivated by water bath at 37 ℃ for 1 h and metal bath at 85 ° C for 10 min to run the gum.
The vector was sequenced with universal primers
① Take 1 tube of BL21 competent cells and place on ice to thaw;
② Per 100.0μl competent cells were added to approximately 2μL of the sequenced successful plasmid, mix by gently flicking the wall of the centrifuge tube (vortex is prohibited), and place on ice for 30 min;
③ Heat shock: place the centrifuge tube in a 42 ° C water bath and heat shock for 60 s, note: do not shake the centrifuge tube;
④ Ice bath: quickly transfer centrifuge tube to ice bath, place 2-3min;
⑤ Resuspend: add 500 µ l per tube of competent cells μL of sterile, antibody free LB liquid medium and incubated at 37 ° C in a shaker at 250 rpm for 1 h with shaking;
⑥ Coating: after centrifugation at 3500 rpm for 5 min. After resuspension of the bacteria, spread the remaining broth evenly all over the LB plate (containing antibiotics);
⑦ Culture: after the coating liquid on the plate was completely absorbed, the culture dish was inverted and the colonies were observed after placing into the 37 ℃ constant temperature incubator for 12 ~ 16h.
① Activated species: single clones were picked and inoculated in LB tubules containing resistance and incubated overnight at 37 ℃ in a shaker at 220 rpm;
② After removing 600μμ l of broth was added to 400μL 50% glycerol, well labeled, and stored at - 80 ℃ in cold water;
③ Transconjugation: 100 µ l of overnight broth was taken to the LB tubules containing the resistance and then incubated at 37 ℃ on a shaker at 220 rpm for approximately 3-4 h;
④ Take 300μL broth is labeled " pre induction ". The remaining broth was cooled at 16 ° C and 220 rpm for 1 h, and then 5μL IPTG (1 M) to induce E. coli to produce the protein of interest. The plates were incubated overnight at 16 ° C at 220 rpm for 12 h. Take 300μL broth is labeled "" post induction "";
⑤ The pre - and postinduction bacterial fluids were centrifuged at 12000 rpm for 1 min, and the supernatant was discarded and the bacteria were added to 200μL 1×PBS was mixed and centrifuged at 12000 rpm for 1min;
⑥ The supernatant was aspirated into a new EP tube, labelled 'supernatant' and the pellet added 200 UL of 1×PBS, mix, and label " pellet "; To the supernatant and precipitate samples, 50 μL of reducing 5×load buffer, mix by gentle shaking, do not invert up and down, and boil the sample, (onto a metal bath set to the conditions, 100℃, 15 min), pending electrophoretic detection.
① Activated species: take 5μL of BL21 glycerobacterium previously preserved with recombinant plasmids, inoculated in LB tubules containing resistance, and incubated overnight at 37℃ in a shaker at 220 rpm;
② Pour the broth into a 500 ml conical containing 250 ml LB broth, add 100μL Kana（100 mg/mL）。The plates were incubated at 37 ℃ at 220 rpm for 5 h in a shaker until the bacterial fluid became turbid at 16 ° C and cooled at 220 rpm for 1 h before adding 250μL IPTG (1 M) to induce E. coli to produce the protein of interest. Overnight incubation at 16 ° C at 220 rpm for 12 h;
③ The broth was centrifuged at 4000 rpm at 4 ° C for 20 min, the supernatant discarded, and the pelleted E. coli were transferred to a beaker with a spoon and resuspended with suspension buffer (20 mm Tris HCl, pH 8.0, 300 mM NaCl) with the addition of lysozyme to give a final concentration of 0.1 mg / ml;
④ The resuspended bacteria were disrupted by ultrasonic disruptor on ice for 15 min and centrifuged at 18000 rpm for 30 min at 4 ° C using a high-speed centrifuge. After the supernatant was incubated with nickel medium for 1.5 h at 4 ° C, the proteins were washed away with wash buffer (20 mm Tris HCl, pH 8, 300 mM NaCl, 10 mm). The protein of interest was eluted with elution buffer (20 mm Tris HCl, pH 8, 300 mM NaCl, 100 mm). All samples were blue stained with G250 during elution of eluted hybrids;
⑤ The eluted protein solution was contained in a 10 kDa enriched tube, centrifuged at 4 ° C, 3400 RPM in a centrifuge, and replaced with suspension buffer (20 mm Tris HCl, pH 8, 300 mM NaCl) to yield a 1 ml protein solution of Tris buffer system containing 50 mM NaCl;
⑥ The protein concentration was determined by UV spectrophotometer at 280 nm.
A 1 ml solution of purified protein in 20 mm Tris HCl, pH 8, 300 mM NaCl was mixed with 6mm PET in an EP tube.
Configuration volume: 1000 ml
① Weigh: weigh 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl, respectively, in a beaker;
② Solubilization: 700 ml dH2O was added in a beaker and stirred with a glass rod so that the powder was completely dissolved;
③ Bring to volume: pour the solution into a measuring cylinder and add water to 1000 ml
④ Aliquot: aliquot the medium to a small tube (5 ml / tube)
⑤ Sterilization: sterilize at 121 ℃ for 20 min.
LB + 0.5m sorbitol + 0.38M mannitol
cotb-mlcc-F: (gene specific primer TM calculated as: 60.7 ° C homology arm length: 15 BP homology arm GC content: 66.7% digestion site: aagctt)
5'- caggaggcgcaactcaagcttTTGCCATGAGCAAGAGGAGAA -3'
cotg-mlcc-F: (gene specific primer TM calculated as: 62.9 ° C homology arm length: 15 BP homology arm GC content: 66.7% digestion site: aagctt)
5'- caggaggcgcaactcaagcttTTGCCATGGGCCACTATTCC -3'
cotx-mlcc-F: (gene specific primer TM calculated as: 62.1c homology arm length: 15 BP homology arm GC content: 66.7% digestion site: aagctt)
5'- caggaggcgcaactcaagcttTTGCCATGGAAAGCAGACCA -3'
cotc-mlcc-F: (gene specific primer TM calculated as: 60.3 ° C homology arm length: 15 BP homology arm GC content: 66.7% digestion site: aagctt)
5'- caggaggcgcaactcaagcttTTGCCATGGGTTATTACAAAAAAT -3'
cot-mlcc-R: (gene specific primer TM calculated as: 60.8c homology arm length: 15 BP homology arm GC content: 66.7% digestion site: aagctt)
5'- ccgagctcgaggcaaaagcttTTACTTATCGTCGTCATCCTTGTAATC -3'
Primer processing: Centrifugation was performed at 12000 rpm for 10 min, and primers were diluted to 10 × by pressing the message on the walls of primer tubes plus ddH2O μM final concentration.
The basic components of the PCR reaction include: template DNA (band amplified DNA), primers, 4 deoxyribonucleoside triphosphates (dNTPs), DNA polymerase, and appropriate buffers.
PCR consists of three basic reaction steps of denaturation annealing extension:
① High temperature denaturation of template DNA: after the template DNA is heated to about 94 ℃ for a certain time, the template DNA double strand is unwinded, become a single strand, so that it binds to the primer, for the next round reaction preparation;
② Low temperature annealing (renaturation) of template DNA to primers: after denaturation of template DNA into single strands by heating, the temperature is reduced to about 55 ℃, and the primers bind to the complementary sequence of the single strand of template DNA paired;
Isothermal extension of primers: the DNA strand complementary to the template is synthesized by extension from the 5 'end → 3' end of the primer with dNTPs in the presence of DNA polymerase (optimal activity at about 72 ℃). Each cycle underwent denaturation, annealing, and extension with a 1-fold increase in DNA content.
> Template <0.5μg
> The upstream primer (10μM） 1μl
> The downstream primer (10μM） 1μl
> ten×PFU buffer (containing Mg2 +) 5μl
> dNTP（10mM） 1μl
> PFU DNA polymerase 1.25μl
> ddH2O up to 50μl
> PCR reaction procedure (three-step):
> Pre denatured at 95 ° C for 5 min,
> 30 recycles：95℃ 30 s，60℃ 45 s，72℃ 1kb/2 min
> 72℃ 10 min
> Hold at 4 ℃.
Samples of post amplification DNA products were collected and spiked with 6×The DNA loading buffer was mixed for loading, electrophoresed at 120 V constant voltage for 20 ~ 30 min, and electrophoretic pictures were saved（Agarose gels were prepared by mixing: 1×TAE buffer + agarose, heat in microwave oven until agarose all dissolve then cool to below 60 ℃ add nucleic acid dye mix, 1.5g/100ml agarose, and 20μL/100mL nucleic acid dye）
The fragment of interest was recovered according to the procedure of the agarose gel recovery kit and finally eluted with 30μL ddH2O after heating and stored at - 20 ℃.
Preparation of plasmid DNA: extract the desired carrier plasmid with the column plasmid DNA miniprep Kit (always with lysozyme).
① Vector digestion reaction system (50 µ L):
>ten× Green Buffer 5 µL
> Hind III 2.5 µL
> Plasmids ~ 2.5μg
> DdH2O was made up to 50 µ L
The reaction system was inactivated by water bath at 37 ° C for 1 h and metal bath at 85 ° C for 10 min
② Purification of digested products: the digested products were recovered according to the procedure of Agarose gel recovery Kit and finally digested with 30μL ddH2O elution, store at - 20 ℃.
Using seamless cloning kit to connect the target gene and the plasmids after double digestion, the reaction system was as follows:
Plasmid gene of interest (10 µ L):
> The target DNA fragment yμL
> Vector DNA xμL
> two×Basic Assembly Mix 5 μL
> ddH2O was supplemented to 10μL
10μL reaction system, the vector to individual insert addition is recommended to be between 0.01 and 0.25 pmols, and the optimal molar ratio of vector to individual insert is 1:2.
Pmols = mass ng / (fragment length BP× 0.65 kDA)
As if:
100 ng of a 2000 bp fragment equals 100 / (2000×0.65) of about 0.08 pmols.
100 ng of a 5000 bp fragment equals 100 / (5000×0.65) to about 0.03 pmols.
Also do the positive control cloning reaction system:
> Control Insert 1 μL
> Linearized pUC19 Control Vector 1 μL
> two×Basic Assembly Mix 5 μL
> Nuclease-free Water 3 μL
Mix it gently, 55 ℃ for 10min. After the reaction is finished, immediately place the centrifuge tube on ice to cool for several seconds.
① Take 1 tube of bs168 competent cells and place on ice to thaw;
② Per 50.0μL competent cells were added to approximately 5μL of reconstituted product (the remaining reconstituted product was stored at 4℃, if transformation failed, reconstitution was possible every other day), mix by gently flicking the wall of the centrifuge tube (vortex is prohibited), and leave on ice for 5 min;
③ It was then added to a precooled sterile electric cuvette and electroporated with 1 shock with the following settings: 2.0 kV, 25uf, 200 Ω, duration 4.5-5 MS
④ Immediately after the electric shock was completed, 1 ml of resuscitation medium RM (LB + 0.5m sorbitol + 0.38M mannitol) was added, and after repeated gentle pipetting for several times, the solution was aspirated from the electric cup, transferred to a 1.5ml centrifuge tube and placed at 37℃ in a shaker at 180 rpm for 3 h to resuscitate the culture by shaking
⑤ Centrifuge at 3500 rpm for 5min, discard the supernatant 900ul, gently resuspend the cells, respectively 90ul, 10ul of the resuspended cells were spread evenly into LB plates (kanamycin resistance plate), after the liquid was completely absorbed, the plate was inverted, at 37 ℃ incubator overnight culture.
*Positive clones were identified by PCR
① Single clones were picked and inoculated into LB tubules containing the resistance and cultured overnight;
② The corresponding overnight tubules from each clone were removed and 600μl of broth was added to 400μl 50% glycerol, do well for labeling, and store at - 80 ℃ until cold. The remaining broth was plasmid extracted with the column plasmid DNA miniprep Kit (always with lysozyme), and the PCR was followed by run on identification.
*PCR reaction system (50 µ L):
> Template 1μl
> The upstream primer (10μM） 0.5μl
> The downstream primer (10μM） 0.5μl
> ten×PFU buffer (containing Mg2 +) 2.5μl
> dNTP（10mM） 0.5μl
> PFU DNA polymerase 0.6μl
> ddH2O up to 25μl
PCR reaction procedure (three-step):
> Pre denatured at 95 ° C for 5 min,
> 30 recycles：95℃ 30 s，60℃ 45 s，72℃ 1kb/2 min
> 72℃ 10 min
> Hold at 4 ℃.
*Restriction analysis of positive clones
① Single clones were picked and inoculated into LB tubules containing the resistance and cultured overnight;
② The corresponding overnight tubules from each clone were removed and 600μμ l of broth was added to 400μμ l 50% glycerol, do well for labeling, and store at - 80 ℃ until cold. The remaining broth is plasmid extracted with the column plasmid DNA miniprep Kit (always with lysozyme), double digested and run on to identify.
Vector digestion reaction system (50 µ L):
> 10× Green Buffer 2 µL
> Hind III 1 µL
> Plasmids ~ 1μg
> ddH2O was made up to 20 µ L
The reaction system was inactivated by water bath at 37 ° C for 1 h and metal bath at 85 ° C for 10 min to run the gum.
The vector was sequenced with universal primers.
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