Biotransformation

It contains overall process of Biotranformation in Bacteria, I used in lab.

How to do culture of E. coli?

Steps: -

1. First of all, (switch on the power of Laminar Flow if not on) then clean and sterilize the Laminar Flow (Clean Bench) with ethanol.
2. Burn the lamp.
3. Put all the necessary materials inside the Bench.
4. Sterilize the hands with ethanol.
5. Well label the 15ml tubes clearly.
6. Put the 5-6ml LB media or LB sorbitol media into the labelled tubes with shaking properly.
7. Then add 5-6 µl respective antibiotic into the media containing tubes.
8. After that, carefully scratch the bacteria containing required genes from the plate with the help of autoclave tooth pin.
9. Then insert the media and antibiotic containing respective tube with rubbing properly inside the solution.
10. Remove all the items which was used in LF Bench and switch on UV lamp after completing the work.
11. Close the cover properly and keep the tubes in 37°C shaking incubator for 5-6hrs until full growth of bacteria.

How to do plasmid Isolation?

Steps: -

1. After culture, take out the tubes from shaking incubator and do centrifuge at 4°C at 3000rms machine for 10-15 minutes.
2. Throw out the supernant liquid from the tube and add 300µl solution I then vortex it to mix properly
3. Then add 300µl solution II with shaking gently to get clear solution.
4. After that add 300µl solution III which gives precipitate and can be vortex for a while.
5. Transfer to new Eppendorf tube and do centrifuge for 10 minutes. 6 Then take only clear liquid in new Eppendorf tubes with well labelled.

Pour transparent portion into DNA spin without disturbing upper layer. Centrifuse at 8000 for 3min. Throw out filtration. Add 200ul 70% ethanol. Centrifuse at 8000 rpm 2 min. Transfer tube into new EP tube. Add 50ul TE in tube. Tap and room temperature for 3-5 min. Centrifuge 3000 rpm for 1-2 min.

7. Now add 500µl resin after well shaking to Eppendorf tube where resin (positively charged) bind with DNA as DNA itself is negatively charged due to phosphate groups.
8. Then run the vacuum filtration with well cleaned syringe and column in suspension motor. (Note: - syringe and column are cleaned with distilled water and chilled ethanol properly before using)
9. After that wash with chilled ethanol and do centrifuge for 5 mins to remove all ethanol in small centrifuge machine.
10. Now transfer the column in new well labelled Eppendorf tubes and add 30µl TE buffer in column and leave for 5-10minutes.
11. And do the centrifugation for 3-5 minutes to collect isolated plasmid from resin.
12. Finally, required plasmid is isolated which can be used for the purpose and plasmid is stored in -20°C for a long time.

Further Plasmid is digested to get the size of vector and gene for the confirmation.

How to do digestion?

Steps: -

1. Take 2-3µl plasmid in new well labeled Eppendorf tube and add 4µl TE buffer, 1/1µl required restrictions enzymes and 1µl buffer to make total volume of 10µl.
2. Then keep in water bath of 37°C for 45min.-1hr.
3. Then add 4-5 µl loading buffer and now run in gel-electrophoresis for 30minutes.

Preparation of gel for gel electrophoresis

Steps: -

1. After that weigh 0.8gm Agarose (E) for 100ml and mix with TAE Buffer to make total volume 100ml.
2. Then keep the solution in microvan for 2-3 minutes.
3. Now let the solution cool down and add 5 µl EtBR (Ethidium bromide) for 100ml.
4. Prepare the frame for electrophoresis by washing plate, comb and frame sheet with water then ethanol.
5. Pour the solution into the frame fitted with comb and wait for to freeze the gel for half an hour.
6. Load the 4-5 µl sample and marker of 1Kb after gel is ready and run for 30minutes.
7. Then take carefully ran gel into machine for analysis.

Purification of Gene or vector

After running the gel-electrophoresis, the required band is cut in sterilized condition and chop it with knife and put inside new ET carefully then centrifuge for 2-3 mins before putting resin then shake well and keep the ET with gel in 37degree for 10-12 hrs to dissolve the gel completely but if it is urgent then heat the ET in 65degree water bath for 10-15minutes and run through the plasmid isolation vaccum to get the pure gene or vector by finally eluting with 15-20micro liter TE buffer or autoclaved distilled water.

How to do Transformation of Plasmid DNA?

(Note: - All steps are need to done inside Laminar Flow only.)
Steps: -

1. Culture of E. coli XL-1 Blue or BL-21(host bacteria) in transformation tube using 3-5 ml LB media without antibiotic for 5-7hrs.in shaking incubator.
2. After that, keep in ice for 30minutes.
3. Do centrifuge for 10-15 minutes.
4. Then throw the supernant liquid inside Laminar Flow and mix 5ml, 0.1M CaCl2 chilled solution. (Mix with the same pipette inside Laminar Flow).
5. Again do centrifuge for 10 minutes and repeat the same process as step 4. (Washing by 0.1M chilled CaCl2)
6. Then throw the supernant liquid after centrifuge and add 300-400µl 0.1M CaCl2 in the tube and re-suspense by shaking. Now the competent cell is ready for Plasmid Transformation.
7. Add 1-2µl plasmid if concentration is high but if the plasmid is ligation mixture or low concentration then add 10-15µl plasmid with the Competent BL-21 cells.
8. Then keep the mixture in ice for 1-2minutes.
9. After that do heat shock at 42°C for 50sec only.
10. Then keep the mixture in Ice again after the heat shock for 2-3minutes.
11. Then add 300µl LB media inside the Laminar Flow and do the culture by keeping in Shaking Incubator for 30-60minutes at 37°C.
12. Now do plating in respective antibiotic plate with the help of sterilized metal spreader inside the Laminar flow with carefully and keep the plated plate in 37°C Incubator (not shaking Incubator)
13. After the growth of colony (5-7hrs), pick the selected some colony and do the culture again for plasmid isolation to confirm the required your gene then if confirmed, then transfer to the required colony to new plate.

How to do protein expression?

Steps: -

1. First of all, do seed culture by putting 5ml LB media and 5µl respective antibiotic in 15ml tube and keep in shaking Incubator for 5-6hrs.
2. After that when seed is grown, then transfer it to autoclaved conical flask by putting 100ml LB media or LB sorbitol and 100µl respective antibiotic inside Laminar Flow and keep in 37°C shaking incubator.
3. Then after 4-5hrs, if the OD (Optical Density) is around 0.5OD, induction is done by adding IPTG having concentration 0.4mM in the mixture. (Volume of IPTG can be calculated by using the formula M1V1=M2V2)
4. After adding IPTG inside the Laminar Flow, transfer the conical flask into 20°C Shaking Incubator for 18-20hrs.
5. Take out the growth cells and transfer into 50ml tube with well labeled and put tubes always in ice. (Protein must be handled in Ice only.)
6. Now do centrifuge at 4°C.
7. Throw out the supernant solution and the residue is washed with 5-10ml Protein washing buffer (50mM Tris-Cl + 10% Glycerol + 1%NaCl). (Protein washing buffer must be kept in ice only)
8. Now vortex the residue with washing buffer just to mix only not more.
9. Then again do centrifuge for 10minutes and throw out supernant solution and add 5-10ml Protein Washing Buffer and do vortex and centrifuge for 10minutes.
10. After that, throw out supernant solution and add 1ml protein washing Buffer and store at -20°C freeze for long time store.
11. But for the immediate use and checking of the expression of required protein, cell walls are broken by Sonicator.

How to break cells in Sonicator?

Steps: -

1. On the power bottom of the Sonicator.
2. Wash the rod by distilled water and wipe it by tissue paper before use.
3. Adjust the tube containing cells with the rod appropriately (carefully) just at the center without touching the wall of tube.
4. Do all the process in ice at the bottom of the tube always.
5. Set the time for 8-10minutes for 1 tube.
6. Then close the door and run the machine properly.
7. After this, pour the solution in well labelled ET (Eppendorf tube).
8. Then do centrifuge in small centrifuge machine for 30minutes.
9. Then soluble layers of protein is poured into new well labelled ET.
10. For the insoluble part of protein, first mix with 500µl 10% SDS Solution or lysis buffer with properly shaking and then keep in hot water (100°C) for 15 -20 minutes to dissolve. Now for the analysis of required protein, SDS PAGE is carried out.

How to prepare the sample for the SDS PAGE?

Steps; -

1. Take 50µl sample in well labeled ET.
2. Add 30 µl 10% SDS solution or lysis buffer into the ET.
3. Add 20µl PLB (Protein loading Buffer).
4. Then wrap the mouth of the ET containing sample with Parafin Tape or aluminium foil and Clipped in rubber pad.
5. Boil at 100°C water for 25-30 minutes.
6. SDS gel is prepared by the following steps:-
   I. Wash the pates (glass) with distilled water or soap if it is dirty.
   II. Then wash it with acetone.
   III. And dry the glass plates by hair dryer.
   IV. Then make the frame properly by putting glass plates, rubber and clamp carefully.
   V. After that, check the leakage by putting water carefully. If there is no leakage, then keep running gel by removing water with the help of tissue paper.
   VI. Running gel and stacking gel are prepared by mixing the 30% acrylamide/0.8% bisacrylamide , 4× tris-Cl/SDS/PH8.8(running gel)/PH6.8(stacking gel), water, 10% APS(ammonium persulphate ) and TEMED(Tetramethyl-ethylene diamine), mentioned in the protocol according to the size of plate as;-

Stock to add(ml)	10×8cm (1)	10×8cm (1)	10×8cm (2)	10×8cm (2)	10×12cm (1)	10×12cm (1)	10×12cm (2)	10×12cm (2)
------------------	Run	Stac.	Run	Stac.	Run	Stac.	Run	Stac.
30%acrylamide/0.8%bisacrylamde	2.25	0.162	4.5	0.325	3	0.325	6	0.65
4× Tris-Cl/SDS/pH8.8/pH6.8	1.406	0.312	2.812	0.625	1.875	0.625	3.75	1.25
Water	1.946	0.762	3.936	1.525	2.625	1.525	5.25	3.05
10%anmmonium persulphate(µL)	18	6.25	36	12.5	25	12.5	50	25
TEMED(µL)	4	1.25	8	2.5	5	5	10	10

8. After the preparation of gel, put the plate containing gel in the big Cascade (Tank) by removing comb and other unnecessary thing.

9. Add buffer just to cover the well of gel and carefully load 6-7µl prepared samples and marker into the well.

10. Switch on the power and set the time and then press the run bottom.

11. Run the process till all the blue stain crosses the lower bottom of the gel.

12. Now stop the process and transfer the gel into cleaned small trough (bucket) carefully for staining.

13. Then add staining solution and keep for 30minutes -1hrs and remove the staining solution and wash with washing solution I for 45 minutes -1hr and then throw the washing solution I and add washing solution II for 10-15hrs in shaking machine.

14. After that carefully transfer the gel in thin white plastic and dry it for 10-12 hrs and now gel is ready for further analytical use.

Note; - Preparation of staining Solution

1. Brilliant blue_0.25gm
2. Methanol-50ml
3. Acetic acid-10ml
4. Water-40ml.

Preparation of electrophoresis buffer

1. Glycine-7.2gm
2. SDS-0.5gm
3. Tris-Cl-1.5gm Preparation of Washing Solution-I
4. Methanol-50ml
5. Acetic acid-10ml
6. Water-40ml Preparation of solution –II
7. Methanol -10ml
8. Acetic acid-10ml
9. Water-80ml

How PCR is done?

1. First of all, collect the required primer of FP (forward primer) and RP (reverse primer) –from Genoteche or market.
2. After that dilute the FP and RP by TE Buffer taking the mention volume in primer details slip (paper0.
3. Then, dilute further 10 times by TE buffer to make working stock.
4. Collect dNTP( dATP,dTTP, dCTP,dGTP) ,Primex (PCR Primix), DNA template , or template DNA, Primer FP,Primer RP,DMSO,TE buffer or water (better water). Or, we can use pfu primstaer whcich is more efficient. For Primstar pfu:- 2×buffer (5X or 10X) = 25µl DNA template= 2µl Primer, FP = 4µl Primer, RP = 4µl DMSO = 2.5µl TE buffer /water = 9.5µl DNA polymerase = 0.5µl DNTP = 4µl Total volume of mixture = 50µl
5. All these are mixed in PCR tubes then centrifuge it for few minutes and keep in PCR machine after making proper program in PCR Machine. (By checking the appropriate melting temperature of the Primer form the detail slip). Note; - always make 4 types of sample so that optimized conditions can be obtained.
6. After the completion of the PCR run, check the band in gel electrophoresis. (just run 2µl)
7. And do purification if normal gel electrophoresis is ok, make cutting gel and run with all the appropriate PCR product and cut the gel by cleaning the machine and knife with ethanol and cut required portion or part and make small pieces after separating other, then collect pices gel in new Eppendorf tube and add 1ml resin kept in 37°C incubator. And shake up and down for 5-10minutes to dissolve all the gel.
8. Purification by DNA Column like plasmid isolation.
9. After using TE buffer (20-25µl) leave for few minutes and collect the sample by centrifugation but before adding the TE buffer also do centrifugation to remove all the ethanol for 30 minutes.
10. Then do ligation in XL1-Blue host.

For Ligation: -

Steps: -

1. First of all, make all required gene and vector.
2. Then in clean ET, add required gene 3-5µl depending upon concentration of gene, the add vector 3-4µl, ligase 1µl,10× ligase buffer 1µl and TE buffer 2µl making total volume 10µl.
3. First only mix vector and gene, do centrifuge for shor time and keep at 70ºC for 3-5 mins, and put in ice for2-3 min and then add ligase buffer and ligase and mix properly and do short centrifuge and keep the mixture in 16ºC for 16hrse keep the mixture for overnight at 16ºC.
4. Next day ,make ready for competent Xl-1 blue for transformation with ligated gene.as follows:-
   I. First of all, XL1 Blue host is cultured for 5-7hrs in transformation tube at 37°C in LB media without using antibiotic.
   II. Keep in ice for 30minutes.
   III. Centrifuge at 3000rmp for 10’.
   IV. Throw supernatant and take cells only.
   V. Wash the cells with 0.1M chilled CaCl2 (5ml) and wait for 10’.
   VI. Vortex, wait, keep in ice for 10’.
   VII. Repeat the process twice.
   VIII. Centrifuge and take the cells only.
   IX. Add 300-400µl of 0.1M CaCl2 and re-suspend the cells to make ready competent cells of host (XL1 Blue) for ligation.
   X. Then mix the DNA plasmid for 3-5’ and keep in ice. ( ligation mixture is 10-15µl in volume is ok, where plasmid is around 1-2 µl or sometime 3µl if concentration is low)
   XI. Give heat shock at 42°C for 50sec.
   XII. Then keep in ice (2-3)’.
   XIII. Add 300µl LB broth by pipette.
   XIV. Keep the tubes at 37°C for 30-60minutes.
   XV. Do plating in respective antibiotic containing LB plate.
   

How to plate after ligation?

Steps: -

1. Take ampicillin LB plate.
2. Mix 5µl IPTG and 45µl Xgal (from freeze) in ET and transfer to plate.
3. Leave for 10 min in Laminar Bench.
4. Then transformation in above plate by using ligated PCR products grown in 1-2ml LB media (Do proper plating).
5. Incubate overnight.
6. Pick only white colony in fresh LB/amp Plate.
7. After that when colony is grown in new plate, then do culture from each marked colony from the plate and do plasmid isolation and digestion for the confirmation of required vector and gene band in XL1 Blue host.
8. If the proper or correct band is obtained which conforms the proper ligation and the ligated products are sent for gene sequencing. (Sample is sent)

In Vivo reaction (Biotransformation)

1. Prepare seed culture 5-7ml in 15 ml tubes with using appropriate antibiotic.
2. Than after 4-6 hrs later when seed is well grow, transfer the 2-3ml cultured seed to new autoclaved conical flask and keep in 37°C shaking incubator.
3. When OD is 0.6OD at 600nm after around 5-7hrs, add IPTG making final concentration of 0.04mMol.
4. For checking, after about 20hrs, transfer in 50ml tube inside Leminar Flow (sterile condition), then centrifuge cells at 3000rpm for 10minutes.
5. Throw supernant and take cells and add sterile water 30-50ml, 10% glucose (10-16) ml and glycerol 4ml for 50ml total volume.
6. After that mix gently in 15ml tube times 10tubes (according to substrates).
7. Add substrate 0.2mM (200µM) in each tubes ie 20µl.
8. Incubate at 37°C (20-37°C) for 24 to 48hrs.
9. After feeding, reaction sample can be taken at 3hr, 6hrs, 12, hrs, 24hrs, so on to check the production of product by HPLC.
10. For large production, directly feed substrate in to the induced culture after putting IPTG when the cells are grown well after around 16-18hrs.
11. Also add 10% glucose along with substrate of 0.2mM.
12. Then take the sample in 3, 6, 12, 24, 48,72hrs for HPLC analysis and if at the time of checking HPLC, the substrate is found to finish up, then add additional substrate of 0.2mM as previous and so on according to necessity.
13. When the reaction is completed (48-72hrs) then add ethyl acetate twice the volume of the reaction mixture and do centrifuge to collect supernant only in 5ml tube and then keep in drier with cap open to dry completely.
14. Then add methanol and do vertex for 30-60minutes and do centrifuge and collect pure sample for HPLC or further analysis.

Protein purification: -

1. First of all, take the resin by well shaking from its container and then centrifuge it for 10 minutes at 4000 to 4500rpm and throw supernatant.
2. And mixed the resin with equilibrium buffer with the ratio 1:2 to 1:3(resin by volume and vortex well and centrifuge it at 4000 to 4500 rpm for 10 minutes and throw supernatant again.
3. Take soluble protein and mix with washed resin in the ratio 1:10 to 1:20 (resin: soluble protein). Then it shakes gently for 1-2 hours so that all proteins get bind with resin.
4. After that centrifuge the mixed protein resin for 10-15minutes at 4000-4500 rpm and throw supernatant and pour the protein-resin into the well washed purifying column.
5. First wash protein -resin with 10mM imidazole elution buffer or washing buffer(3-5ml) and then elute with 2ml of 50mM, 100, 150, 200, 250, 300mM Imidazole and collect fraction of protein with each concentration of Imidazole.
6. Prepare SDS-PAGE gel and run each sample to check purity.
7. Take pure fractions and concentrate using centricon.→ put all pure fractions → centrifuge → [wash with equilibrium buffer (2ml) → take out concentrate protein.] This steps can be repeated.

How to clean centricon?

a. First of all, put equilibrium buffer, shake well and throw it. b. Again add equilibrium buffer, centrifuge and throw it. c. Add 70% ethanol, shake it and centrifuge and throw and add again. d. Clean all. e. Add 70% ethanol (full of tube) and store at 4°C.

How to re-use it?

1. Throw ethanol and wash it with equilibrium buffer/washing buffer. (twice).
2. Centrifuge and take out centricon and throw the liquid at the bottom.
3. Repeat the process.
4. Add pure fraction of the protein all.
5. Do centrifuge.
6. Throw supernantant at the bottom.
7. Repeat until it is concentrated i.e it reached below 250.

Media preparation:-

LB media:

1. 10gm LB broath per 400ml
2. Distilled water upto the mark
3. Mix with stirrer and autoclaved. LB Sorbital
4. 10gm LB Broath per 400ml
5. 72gm D-Sorbital per 400ml
6. Distilled water up to the mark
7. Betaine – 0.117gm per 400ml
8. Mix properly with stirrer and autoclaved LB Sorbitol in 1 litere
9. 25gm LB Broath per 1000ml
10. 180gm D-Sorbital per 1000ml
11. Distilled water up to the mark
12. Betaine – 0.2925gm per 1000ml
13. Mix properly with stirrer and autoclaved

M-9 media

Solution preparation for plasmid Isolation: -

Solution-I

1. 2M-tirs-HCl (7.5) – 2.5ml/400ml
2. 0.5ml EDTA(pH-8)-2ml/400ml
3. Add 5% glucose (optional)/400ml
4. Add distilled water up to the mark and autoclaved
5. Add RNAse 200µl after autoclaved and cooled solution.

Solution-II

1. 5M NaOH- 4ml /100ml
2. 10% SDS(pH-7)-10ml/100ml
3. Add distilled water and autoclaved.

Solution-III

1. Potassium acetate -12.96gm/100ml
2. Add distilled water making the volume half than 100ml and adjust pH 4.8 by adding acetic acid (glacial acetic acid) and then the volume of the solution up to the mark
3. Autoclaved

T E buffer: -

1. 5ml 2M tris-HCL
2. 4ml EDTA
3. Total volume 100ml
4. Autoclaved

Equilibrium Buffer (100ml)

1. 50mM Tris-HCl= 2.5ml
2. 300mM Nacl- 1.753gm
3. 10% glycerine= 10ml
4. Distilled water

Imidazole (1M) 25ml

1. 1.702gm take the weight of Imidazole and dissolve in equilibrium buffer. BHI plate
2. 15gm BHI media/400ml + 0.9% sorbotal (do autoclave 15mins only but separatelyt as usual )
3. 8gm Baterial agar /400ml