Prepare competent cells 1) Activation of bacteria: 5 mL LB was prepared the night before without antibiotics, inoculated at 1:100, and incubated overnight (less than 16 hours) at 37 ° C. At the same time, a tube of antibiotics was added to see whether bacteria grew (let bacteria revive and enter the early and middle stages of logarithmic phase). It's easier to make competent cells 2) The next morning, transfer it into 100mL medium with a density of 1:2000-500 and culture for 2h (the naked eye can see slightly cloudy, or put the palm on the bottom of the bottle, the palm lines are blurred, rather than too low density culture) 3) Prepare ice boxes and vertically insert multiple 50mL centrifugal tubes for pre-cooling. In the ultra-clean platform, the bacteria liquid was transferred to a 50m pre-cooled centrifugal tube for ice bath for 20min. Then, centrifugation was performed at 4000 RPM at 4℃ for 10 min. 4) After centrifugation, the supernatant was poured out of the ultra-clean platform, and 5mL of pre-cooled 0.1-m cacl2-MgCl2 solution was added to each tube with repeated blowing and suction with the head of a gun. The mud was re-suspended and placed in ice for 10min, followed by centrifugation at 4000 RPM at 4℃ for 10min. 5) After centrifugation, pour off the supernatant in the ultra-clean platform, add 2mL of pre-cooled 0.1m cacl2-glycerin solution to each tube, blow and suck it repeatedly with the head of a gun, re-suspend the mud, and put it in ice for 10min 6) after the ice bath, divide the competent cells into 1.5mL EP tubes, 100uL for each tube, and immediately put the tubes on ice. 7) End of packing. The competent cells were placed in a ziplocked bag, labeled and stored in a refrigerator at -80℃.
Calculate the dosage of various drugs Ampicillin: 0.1g/ mL 5ml (refrigerated) IPTG: 24mg/ml=100mM 5ml (refrigerated) M63 5× (refrigerated, not sterilized for use) : 500ml LB culture medium and LB culture gene autoclave are faulty and need to be re-matched
The following systems are prepared: LB liquid medium 500ml (sterilized, refrigerated after use) LB solid medium 250ml (sterilized), add 250U ampicillin, find the medium solidified, microwave heating to melt again, add 215U ampicillin, flip the plate again, select the best two plates reserved. M63 1x 400ml (sterilized, purchased a pack of powder and added water to 400ml, no glucose, ampicillin added) Glycerin 50% (stored at room temperature) sterilization (120℃, 20min) Thawing of receptive state and plasmid (CP MFP) 3U plasmids were transferred into 5α competent cells, respectively, and coated on the above reserved LB plate. The cells were placed upside down overnight at 37℃ and cultured in a biochemical incubator
Check the culture medium, MFP bacteria colony is not obvious, so it is reformulated LB 250mL was prepared and AGAR 3.75g was added Autoclave for 20min MgSO4 1000X (0.6g 5ml) Preparation of glucose solution 100x (2g 10ml) Amp package added upside down on ultra-clean table (stored upside down in refrigerator) Pick bacteria, add to 3ml LB, add to shaker culture overnight Again, the plasmid is transferred into the receptor cell, They were coated on the above-mentioned reserved LB plates containing Amp and placed upside down overnight at 37℃ to be cultured in a biochemical incubator
Observe the plate in culture, and observe the blue (small) and white (large) colonies in the receptive state of CP. Many colonies were observed on plates transferred to MFP 1L and 100mL of TAE 1x were prepared 50x tae 20ml+ddH2O980ml 50x tae 2ml+ddH2O98ml Find the single colony suitable for picking bacteria and mark it. The labeled colonies were lightly picked on the surface of the culture medium with the tip of a small gun in the ultra-clean stage. The tip of the gun was injected into 500 µ l of LB medium containing Amp. Put them into the shaker for 3h culture (two CP [one white and one blue marked], one MFP {no label, marked as 0}) Later, five colonies (numbered as CP1-5 MFP1-5) were selected from CP (all blue colonies) and MFP respectively, and 500U LB culture medium containing Amp was injected. Incubate in a shaker for 8 hours Take 1μL of each of the two activated bacterial solutions (CP Blue 0 MFP0), add reagents according to the table (error: unsterilized spear head was used), conduct PCR, and set the parameters as follows: 94℃ pre-denaturation for 4min, 94℃ denaturation for 30s, 55℃ annealing for 30s, 72℃ extension for 55s, denaturation, annealing and extension step cycle 30 times; Finally, it was heated for 5min at 72℃ and cooled to 16 ℃.
Preparation of agarose gel solution :(1%30ml 0.3g agarose and 30ml 1xtae) turn the microwave oven to medium-high fire, boil the solution for 7 times during the process (observe before microwave when heating, take out and shake well once boiling, then put back to heat), the solution from cloudy to clear, add 3uDNA dye, shake well, and pour into the gelatinizing tank for solidification (20min). Note: add a little more water (about 1ml) to the configuration, because boiling will cause water evaporation and loss of error 16:20 Electrophoresis: DNA Marker 5uL CP and MFP bacterial liquid PCR samples 8uL each 5μL of 2K DNA Marker (1KB DNA Ladder) and 8μL of PCR products were successively added into the upper sample Wells of the prepared agarose gel, and then electrophoresis was performed at 120V for 40min. (Negative pole - positive pole)
The exposure time of uv observation was 800ms Then 30uL +lb3mL was inoculated at a ratio of 1:100 and cultured in a 50mL centrifuge tube. Cp0 plus IPTG inducer (100mM)15uL (final concentration should be 0.5mm), MFP no.5. Put them together in 210RPM shaker and incubate overnight at 37℃. Bacteria-preserving CP and MFP were stored with 470uL glycerol (1:1) in a refrigerator at -20℃
•The CP (+IPTG) of the night was taken out for observation and no obvious characterization was found. Cp1, which was later selected for insurance, had obvious blue color, while others had vague blue color •Cp (+IPTG) was observed after long standing, and the sinking mud was white. And cp1 fungus mud is blue. •Overnight MFP bacteria were inoculated at 1:100 on M63 medium (20mlM63 20.2umGSO4 20.4U glucose 20.7AMP 200U bacteria; Iptg101.8u) (served in petri dishes) •Repeat the previous step to configure 3 centrifuges packed in 50ml tubes (placed in biochemical incubator without shock culture) •Set two blank controls (without IPTG, cultured in a petri dish in a biochemical incubator) •Develop 24-hour tomorrowwatch In order to explore whether CP white colonies are the bacteria we need, repeat PCR of white colonies (compared with MFP) and run glue (130V 30min) [5000KB marker]
Exposure time: 1000ms It was observed that the PCR results of CP white and CP blue were consistent, indicating different reasons to be analyzed Cp3, 4, 0(white) and MFP1, 2 were 1:1 added with 50% glycerol and put into -20℃ refrigerator to preserve bacteria Cp1, 2, 5 and MFP3, 4, 5 in 4℃ refrigerator (reduce activity)
It was observed that CP0 white, CP3 and CP4 all showed blue characterization. MFP (M63) was relatively clear and had no obvious characterization 50mL 1M HCl was mixed with 2.4m HCl With the 1 m NaOH 0.1wt/vol crystal violet solution -0.1g crystal violet + 100mL water
In the morning,the bacteria in the cp large test tube did not have blue characterization, while the bacteria in the small centrifuge tube had blue characterization The petri dish of MFP is vaguely sticky. Start double digestion. With two double digestion systems:
Enzymatic digestion at 37 ° for 20min on PCR instrument Run electrophoresis 130V 30min+10min (using 5KB marker) add loading buffer!! (After loading buffer is added, it is pumped evenly with a gun)
There should be two strips in the left column that do not meet expectations But still cut the glue Plastic recycling: The colloidal CP glue weight 0.044g MFP glue weight 0.04g Add PE sol solution (triploid product, glue 0.1g as 100U) CP134U MFP120U metal bath at 25℃ for 10min 25mlPW+ anhydrous ethanol solution The solution was added to the CA5 adsorption column (the adsorption column was placed in the collection tube) and stood at room temperature for 2min Centrifuged at 12000rpm for 45s, the waste liquid was dumped and the adsorption column was put into the collection tube Add 600uPW+ anhydrous ethanol, centrifuge at 12000rpm for 45s to drain the waste liquid, and put the adsorption column into the collection tube (twice) Centrifuge at 12000rpm for 2min, and dry Change the centrifugal tube, add buffer TB50ul to the adsorption film, place at room temperature for 2min, centrifuge at 12000rpm for 2min, collect DNA solution Experimental results: The plasmid containing MFP gene ran gel electrophoresis results only one strip To verify whether the results do not meet expectations due to experimental operation errors Double digestion was carried out again, and the total system was 50U according to the instructions Enzymatic digestion at 37 ° for 20min on PCR instrument
The result is the same as last time... Analysis may be the enzyme inactivation or plasmid restriction site problems (dangerous (# 'O')) The cut glue is stored in the refrigerator at 4℃ Contacted Dr. Wang Yanyi to ask about MFP biofilm, and decided to take a petri dish for crystal violet staining tomorrow
Recycle the glue again like yesterday's steps (stored at 4℃ yesterday) Plastic recycling: The colloidal CP glue weight 0.044g MFP glue weight 0.04g Add PE sol solution (triploid product, glue 0.1g as 100U) CP134U MFP120U metal bath at 25℃ for 10min 25mlPW+ anhydrous ethanol solution The solution was added to the CA5 adsorption column (the adsorption column was placed in the collection tube) and stood at room temperature for 2min Centrifuged at 12000rpm for 45s, the waste liquid was dumped and the adsorption column was put into the collection tube Add 600uPW+ anhydrous ethanol, centrifuge at 12000rpm for 45s to drain the waste liquid, and put the adsorption column into the collection tube (twice) Centrifuge at 12000rpm for 2min, and dry Change the centrifuge tube, add buffer TB50ul to the adsorption film, place at room temperature for 2min, centrifuge at 12000rpm for 2min, collect DNA solution [The blue mark on the side wall of the centrifuge tube is 2, CP6ng /uL mFP7.6ng /uL, store in the refrigerator at -20℃] 1.Single digestion verification (verify whether the enzyme is inactivated) With 2 enzyme digestion systems:
(Add 2μL 6×loading buffer when running gel)
(Add 2μL 6×loading buffer when running gel) Enzymatic digestion at 37 ° for 20min on PCR instrument
Note: Don't forget to add plasmids ！！！！！！！！！！ (; '⌒ `) 2.Crystal violet staining test Remove supernatant and rinse 3 times with 3mlddH2O +CV solution 10mL staining for 15min When the dye +ddH2O was poured out for several times, the bottom layer of the medium could not be rinsing, indicating that mucin was formed and mineralization could begin
3:00 PM SBF mineralization One petri dish with and without IPTG was reserved as the control, and the rest of the supernatant was poured out and added with about 11mL SBF solution. Culture in biochemical incubator at 37℃. Run electrophoresis 130V 30min+10min（5kb marker）
In the morning Single enzyme 1 h Run glue
In the afternoon The system with double digestion is as follows
run
Plasmid plasmid CP MFP 50ul+5mlLB (Amp included) Cp1 300U +3mlLB (including Amp) Shaker culture at 37℃ overnight Double enzyme cutting glue refrigerator preservation [Date marked]
Plasmid extraction (two tubes of CP and MFP, each with 1ml bacteria) PWT+ 50mL anhydrous ethanol was configured RNaseA and TIANRed were added to P1 1ml bacterial solution 12000rpm 1min, and the supernatant was sucked out Add 150uP1 and re-suspend the fungus mud Add 150uP2 and gently flip it up and down 7 times to change color to clear purple After adding 350uP5, mix it up and down quickly for 12-20 times, turn yellow and flocculent precipitation appears at 12000rpm for 2min Transfer the suction supernatant to adsorption column CP3 (adsorption column into the collection tube) 12000rpm 30sDump the waste liquid, add 300uPWT 30s to the adsorption column, and dump the waste liquid 12000rpm 1min (remove the residual bleaching solution) The adsorption column was placed in a clean collection tube, and eluting buffer TB (CP1, CP, 2 a senior student added 60U MFP1, MFP2WR added 50U) was dropped to the center of the adsorption film for 12000rpm 30s Drop adsorption column Plasmid concentration was measured Cp1 (34.9ng/ul), CP2 (33.2ng/ul), MFP1 (59.4ng/ul), MFP2 (78.6ng/ul)
In the morning MFP PCR system is as follows
pcr
In the afternoon Recycle according to instructions MFP single enzyme digestion (X enzyme)/ double enzyme digestion system
Enzyme digestion at 37 ° for 1h and inactivation at 80 ° for 20min Run rubber 40 min ~ ~ The DNA of morning PCR had no restriction sites Therefore, the energy p can be exchanged to re-pcr the primers of the restriction site 50 ul distribution system
PCR was followed by gel running (one part), purification of the other part and preservation at -20℃ Cut cp again
recycle
Two Gibson systems are prepared
The PCR apparatus was incubated at 50 ℃ for 30min Two tubes of DH 5α receptor cells were co-transformed
With a PCR system (fluorescent protein)
PCR data extension time was changed to 75s With a double digestion system (blue light)
Enzyme digestion was performed at 37℃ in water bath and then transferred to a centrifugal tube for inactivation at 80℃ for 20min in metal bath PCR products and enzyme digestion products were stored in the refrigerator at -20℃ The bacteria selected no. 22 were inoculated at 1:100 (30U bacteria, 3mlLB) and shaken overnight
In SBF Purified yesterday's PCR product Enzyme MFP
The PCR MFP concentration was 50.8ng/ul The cp concentration of glue recovery was 6.2ng/ul The concentration of CP was 23.1ng/ul The T4 connection system is equipped according to the instruction
Store overnight at -20°C Chloramphenicol 25mg/ml 4ml (0.1g chloramphenicol 4ml anhydrous ethanol) was divided into 4 small centrifuge tubes [Note: Chloramphenicol is configured with anhydrous ethanol] Chloramphenicol 1:1000 LB was given Pour on the plate
T7 and GFP plasmids were extracted PCR run
87/5000 Crystal violet staining 8.22 plate of MFP and cp The staining effect of both plates is not good In the afternoon Purify the morning plasmid Mfp SBF pour water and dry at 60°C for 24h Gibson: T7, GFP and Blue light plasmid
T4 connection cp+ MFP, P3K3 Coconversion Colony PCR (Gibson-linked CP + MFP)
Wrong downstream primer!!
Pick out 4 BL+T7 strains 3 CP + MFP strains connected by T4 BL+GFP four, P3K3 three
The bacteria selected yesterday were inoculated with 30U bacteria 3mlLB at 1:100 Take the remaining 200u bacteria 1:1 and add 50% glycerol retaining bacteria into the same ziplocked bag at -20℃ and write down the strains
New primers are in. Re-pcr 11 tube（enzyme cp+mfp1、2、3、4 T4 cp+mfp1、2、3）
Extension time changed to 45s and other unchanged run（marker 胶1、2、3、4 enzyme1、2、3、4 T4 1、2、3）
Enzyme 3 T4 1, 2 and 3 with obvious bands were selected for plasmid extraction extract the plasmid enzyme3 T4 1、2、3 T7+BL1、2、3、4 P3K3 1、2、3
Put the date in the fridge at -20℃
A double anti LB plate was poured into 5 plates and the remaining LB(no antibiotics) was stored at room temperature BL+T7 and CP + MFP co-transformation (BL21 receptor cells have T7 enzyme, can not be used, using the 5α sensible state (without T7)
Recoconversion
Pick the bacteria and number 1① 1② 1③ 2① 2② 2③ 3① 3② 3③ 4① 4② 4③ Wrap the culture in aluminum foil
Bacteria 2② were inoculated at 1:100 for 1.5h at 7:30:100 and inoculated on M63 medium 20mlM63 20.2umGSO4 20.4U glucose 20.7AMP and Cm 200U; Iptg101.8u) blue light irradiation culture
20mlM63 20.2uMgSO4 20.4uGlucose 20.7Amp 20.7uCm 200uBacteria;IPTG101.8u drew 2 pictures with CP
drew another 2 pictures with CP got another M63
CV dyeing
In order to improve the CP element, enzyme digestion is required
Above is the PCR product of CP
Change cp to GFP The fragment was recovered by ligase T4
The strain was cultured overnight in liquid LB medium.
The bacteria solution was added into the fresh LB medium (containing IPTG) at 1:200 and cultured at 220 RPM at 37 ℃ for 8 hours. The 200 µ l culture was transferred to a 96-well plate and the 485/510 fluorescence and OD600 were measured using a microplate reader. The ratio of fluorescence value to OD600 was used to indicate the expression level of sfGFP (vertical axis in the figure below).
Changed the sensor system The cotransferees were connected to two 600U LB (including Amp cm) tubes at 1:100. One tube wrapped in tin foil, one tube not wrapped. Put them together on the shaker. Then connect 4 M63 20mL disks, of which Amp CM glucose and MgSO4 were added 20U each in two disks and Amp glucose and MgSO4 were added 20U each in the other two disks Two plates of CP bacteria were transferred at 1:100, respectively One of the plates of cotransferrable bacteria was wrapped in cut-out paper and illuminated with blue light in a biochemical incubator
M63 was dumped and added to 10mlSBF for mineralization The two tubes were centrifuged 12000rpm for 1min to observe and compare
The left side is not hiding from light and the right side is completely hiding from light
Replace the SBF
Drain the SBF and dry it
Cotransferrable bacteria are on the left and CP bacteria are on the right
