GENE CLONING
CELL GROWTH
ISOBUTANOL
CYANOBACTERIA
ANALYTICAL

GENE CLONING AND EXPRESSION

PCR
  1. TaKaRa Taq™ DNA Polymerase*1 (5 U/μl) 50 μl (250 U)
  2. dNTP Mixture*2 (each 2.5 mM) 1.28 ml
  3. 10X PCR Buffer (Mg2+plus) 1ml 100 mM Tris-HCl (pH 8.9) 500 mM KCl 15 mMMgCl2
  4. 10X PCR Buffer (Mg2+ free) 1 ml 100 mM Tris-HCl (pH 8.9) 500 mM KCl
  5. MgCl2 (25 mM) 1ml
  6. Control Template (1 μg/ml λ DNA) 100 μl
  7. Control Primer 1*3 (20 pmol/μl) 50μl
  8. Control Primer 2*3 (20 pmol/μl) 50μl
  9. Control Primer 3*3 (20 pmol/μl) 50μl
  10. EcoT14 I Marker (100 ng/μl)*4 40μl
  11. 6X Loading Buffer*5 1 ml

A. Amplification using the TaKaRa PCR Thermal Cycler Dice

This kit includes λDNA and primers for amplifying specific target regions of λDNA (6,012 bp or 500 bp).
1.Prepare the PCR reaction mixture in a microtube by combining the following reagents in a total volume of 50 μl.

Reagent Volume Final conc.
10X PCR Buffer (Mg2+ plus)* 5 μl [1X ]
dNTP Mixture 4 μl each 200 μM
Control Primer 1 0.5 μl 0.2 μM
Control Primer 2 or 3 0.5 μl 0.2 μM
TaKaRa Taq 0.25 μl 1.25 U/50 μl
Control Template 0.5 μl 0.5 ng/50 μl
Sterile purified water 39.25 μl
Total 50 μl

* 10X PCR Buffer (Mg2＋ free) and MgCl2 solution may be used instead of 10X
PCR Buffer (Mg2+ plus) if necessary.

2)Place the tubes in a thermal cycler.

3)Perform the reaction under the following conditions.
When amplifying 6,012 bp with Control Primers 1 and 2:

  • 94℃ 1 min (denaturation) 30 cycles
  • 68℃ 4 min (annealing and extension)
  • 72℃ 5 min 1 cycle

When amplifying 500 bp with Control Primers 1 and 3:

  • 94℃ 30 sec (denaturation)
  • 25 cycles 55℃ 30 sec (annealing)
  • 72℃ 30 sec (extension)
  • 72℃ 2 min 1 cycle

B. Amplification of Experimental Samples

The protocol for the samples is basically the same as the control experiment described in A. The parameters of each step (temperature, time) must be optimized for specific DNA templates depending on the size of target, the target sequence, and the length of the primers.

C. Electrophoresis

  1. Remove 5 - 10 μl from each PCR reaction for analysis on an agarose gel, and add ⅙ volume of 6X Loading Buffer to each sample.
  2. Run the samples from Step 1 on an agarose gel. The gel composition and electrophoresis conditions will vary depending on the sizes of the PCR products.
  3. After electrophoresis is complete, stain gels by soaking in SYBR Green I or Ethidium Bromide solution (1 μg/ml) for 20 - 30 min.
  4. Determine the sizes of the PCR products under UV illumination.
AGAROSE GEL ELECTROPHORESIS[2]
  1. Agarose Powder
  2. Ethidium Bromide
  3. 1X TAE (Tri HCL Acetate EDTA)

EQUIPMENTS :

  1. Gel Casting Tray
  2. Well Comb
  3. UV Transilluminator
  4. Electrophoresis
  5. Cell
  6. Voltage cell
  7. Microwave

Preparation 1X Of TAE Buffer:

To prepare 1x TAE Buffer, dilute 20 ml of 50X TAE buffer in 980 ml of distilled water

PROTOCOL :

1. Prepare 1% of Agarose gel by mixing 1g of agarose powder in 100 ml of 1X TAE Buffer.
2. Microwave the mixture for 1-3 minutes till the agarose is completely dissolved giving a clear solution.
3. Let the agarose cool down to palm bearable heat.
3. Add ethidium bromide (EtBr) to a final concentration of approximately 0.2μg/mL.
4. Pour the agarose into a gel tray with the well comb in place.
5.Once solidified, place the agarose gel into the gel box (electrophoresis unit).
6. Fill the gel box with 1xTAE (or TBE) until the gel is covered.
7. Add a loading buffer to each of your DNA samples.
8. Carefully load a molecular weight ladder into the first lane of the gel.
9. Carefully load your samples into the additional wells of the gel.
10.Run the gel at 80-150 V until the dye line is approximately 75-80% of the way down the gel.
11.Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box.
12. Using any device that has UV light, visualize your DNA fragments.
14. Using the DNA ladder in the first lane as a guide , you can infer the size of the DNA in your sample lanes.

PCR CLEAN UP OR GEL ELUTION FROM AGAROSE GEL[3]
  1. NucleoSpinⓇ Gel and PCR Clean-up Kit From TakaraⓇ
  2. 96–100 % ethanol

EQUIPMENTS :

  1. 1.5 mL microcentrifuge tubes
  2. Disposable pipette tips
  3. Manual pipettes
  4. Centrifuge for microcentrifuge tubes
  5. Heating block, water bath, or thermomixer for gel extraction
  6. Scalpel to cut agarose gels
  7. Vortex mixer
  8. Personal protection equipment (lab coat, gloves, goggles)

Preparation of Working Solution of Elution Buffer:

Add 200 ml of 96–100 % ethanol to the given 50 ml wash buffer(NT3) stock solution.

Excise DNA fragment / solubilize gel slice:
  • Take a clean scalpel to excise the DNA fragment from an agarose gel. Remove all excess agarose.
  • Determine the weight of the gel slice and transfer it to a clean tube.
  • For each 100 mg of agarose gel < 2 % add 200 μL Buffer NTI(Be cautious while using the NTI Buffer and wear gloves).
  • Incubate sample for 5–10 min at 50 °C. Vortex the sample briefly every 2–3 min until the gel slice is completely dissolved!

Bind DNA

  • Place a NucleoSpin® Gel and PCR Clean-up Column into a Collection Tube (2 mL) and load up to 700 μL samples.
  • Centrifuge for 30 s at 11,000 x g.
  • Discard flow-through and place the column back into the collection tube.

Wash silica membrane

  • Add 700 μL Buffer NT3 to the NucleoSpin® Gel and PCR Clean-up Column.
  • Centrifuge for 30 s at 11,000 x g.
  • Discard flow-through and place the column back into the collection tube.

Dry silica membrane:

  • Centrifuge for 1 min at 11,000 x g to remove Buffer NT3 completely.
  • Make sure the spin column does not come in contact with the flow-through while removing it from the centrifuge and the collection tube.

  • Place the NucleoSpin® Gel and PCR Clean-up Column into a new 1.5 mL microcentrifuge tube.
  • Add 15–30 μL Buffer NE and incubate at room temperature (18–25 °C) for 1 min.
  • Centrifuge for 1 min at 11,000 x g.
  • Collect the solution in the microfuge tube for further analysis.
PLASMID CONSTRUCTION [12]
  1. Your two Part Samples, A and B: Miniprepped DNA (in BioBrick RFC[10] plasmid backbones)
  2. Linearized Plasmid Backbone (with a different resistance to the plasmid backbones containing your part samples)
  3. EcoRI,PstI
  4. NEB Buffer 2
  5. BSA
  6. dH 20

DIGEST

Enzyme Master Mix for Plasmid Backbone (25ul total, for 5 rxns) Enzyme Master Mix for Part A (25ul total, for 5 rxns) Enzyme Master Mix for Part B (25ul total, for 5 rxns)
5 μl NEB Buffer 2 5 μl NEB Buffer 2 5 μl NEB Buffer 2
0.5 μl BSA 0.5 μl BSA 0.5 μl BSA
0.5 μl EcoRI 0.5 μl EcoRI 0.5 μl XbaI
0.5 μl PstI 0.5 μl PstI 0.5 μl PstI
18 μl dH20 18 μl dH20 18 μl dH20
  • Add 4 μL of linear plasmid backbone (25 ng/μL, 100 ng total) and 4 μL of enzyme master mix to the digested plasmid backbone.
  • Digest Part A and add 4 μL of Part A (25 ng/μL, 100 ng total). Add 4 µl enzyme master mix.
  • Digest Part B and add 4 μL of Part B (25 ng/μL, 100 ng total).
  • Add 4 µl enzyme master mix.
  • Incubate cells for 3 hours at 37

LIGATION

  1. Add 2 µL of digested plasmid backbone (25 ng).
  2. Add an equimolar amount of Part A fragment (digested with EcoRI-PstI) (approximately 3 μl).
  3. Add an equimolar amount of Part B (EcoRI-PstI digested fragment) ( < 3 μL). Add 1 µl T4 DNA ligase buffer.
  4. NOTE: Do not use fast ligase. Add 0.5 μl T4 DNA ligase. Add water to make 10μl. Ligate at 16°C for 30 minutes, heat sterilization 80 °C at 20 minutes Transform with 1-2 μl of product. Incubate the cells at 16℃ for 16 hours.
  5. Transformwith 1-2 μl of product. Incubate the cells at 16℃ for 16 hours.

Note: For linearized plasmid backbones provided by iGEM HQ, a plasmid backbone with an insert of BBa_J04450 was used as template. As a result any red colonies that appear during your ligation may be due to the template as a background. Digesting with Dpn1 before use should reduce this occurrence.

SDS PAGE TO CHECK THE SIZES OF PROTEIN FRAGMENTS[4]
  1. Separating gel mix
  2. 10% SDS
  3. 10% APS
  4. TEMED
  5. Solubilizing buffer
  6. Stainer (Coomassie Brilliant Blue)
  7. Destainer
  8. 1x Running Buffer
  9. 10ul of each Protein sample
  10. Sample solubilizing buffer

Equiments

  1. Glass plates
  2. Microfuge tubes
  3. Pipette
  4. Casting frames
  5. Casting stand
  6. SDS page apparatus
  7. Electrodes
  8. Power supply

Preparation of Working Solution Of APS And Buffer:

APS Working solution - Prepare fresh every time 0.1g of APS in 1ml of water. Running Buffer - Dissolve 1 Pack of running buffer salt fully in 1 L of distilled water.

Procedure

  1. Set the casting frames (clamp two glass plates in the casting frames) on the casting stand.
  2. Prepare the separating gel mix as follows in a separate small beaker:
    • Separating gel mix- 8 ml
    • 10% SDS - 80 Microliters
    • 10% APS- 80 microliters
    • TEMED - 8 Microliter.
  3. Swirl the solution gently but thoroughly.
  4. Pipette of separating gel solution into the gap between the glass plates.
  5. Make the top of the separating gel horizontal, fill in water (either isopropanol) into the gap until an overflow.
  6. Wait for 20-30 min to let it gelate.
  7. Discard the water left above the gel and you can see the separating gel left.
  8. Prepare the stacking gel mix as follows in a separate small beaker:

Sample Preparation

  1. Label the Microfuge tubes as I, II, III.
  2. Take 10ul of each Protein samplec to the respective tubes.
  3. Add 10ul of sample solubilizing buffer to each tube and heat at 100°C for 3 to 5 min.

Preparation of Running buffer

Prepare 1X Running Buffer by dissolving 1Pack of running buffer salt fully in 1L of distilled water and mix well.

Running of Electrophoresis

  1. Make sure of a complete gelation of the stacking gel and take out the comb. Take the glass plates out of the casting frame and set them in the running buffer dam.
  2. Pour the running buffer into the required level in the buffer chambers.
  3. Connect the electrodes with the power supply. Load prepared samples as such into wells and make sure not to overflow. Load 20ul of protein marker into the separate lane.
  4. Set volt at 50V till the dye reaches the separating gel and increase the volt to 120V Turn off the power supply while the dye reaches the footage separating gel.
  5. Carefully remove the gel from the setup and wash with water.

Staining and Destaining

  1. Add Stainer (Coomassie Brilliant Blue) till the gel gets immersed for 30 minutes on a rocking table (can be reused).
  2. Add destainer till the gel gets immersed (can be reused), Store the used destainer in a separate bottle.
  3. De-stain the gel till it gets a clear background.
  4. Remove de-staining solution, rinse gels twice with distilled water.
  5. Store the gel in distilled water

CELL GROWTH AND METABOLISM

COMPETENT CELL PREPARATION[5]
  1. LB broth.
  2. Ice-cold
  3. MgCl2 solution.
  4. Ice cold CaCl2 solution.
  5. Chloramphenicol.
  6. Distilled Water.

Equiments

  1. Conical flask
  2. Ice-cold falcon tubes
  3. Centrifuge tubes
  4. Colorimeter Centrifuge.

Procedure

  1. Inoculate a loopful of E.coli strain onto 100 ml of LB broth.
  2. Incubate at 37°C with agitation until the culture reaches 0.6 OD.
  3. Chill the culture in ice for 30 minutes.
  4. Aliquot 1.5 ml of culture into 2 sterile microfuge tubes using sterile micropipette tips.
  5. Centrifuge at 4000 rpm for 10 minutes at 4°C.
  6. Remove the supernatant by aspiration with pipette and resuspend the pellet with 300μL of ice cold calcium chloride solution. Incubate the cells in ice for 15 minutes. Centrifuge at 4000 rpm for 10 minutes at 4°C. Remove the supernatant by aspiration with pipette and resuspend the pellet with 700μL of ice cold calcium chloride solution and incubate the cells in ice for 30 minutes. Centrifuge at 4000 rpm for 10 minutes at 4°C.
  7. Remove the supernatant by aspiration. For long term storage of competence cells resuspend the pellet in 100μL CaCl2+glycerol mix, stored at -20°C/4°C.

Transformation

  1. Take 200 l of the above cell suspension in two 2.0 ml collection tubes and label them as ‘control’ and ‘transformed’. Add 2 l of plasmid DNA to the tube labeled as transformed and mix well.
  2. Incubate both the tubes on ice for 30 minutes
  3. Transfer them to a preheated water bath set at a temperature of 42oC for 2 minutes (heat shock).
  4. Rapidly transfer the tubes on ice-bath. Allow the cells to chill for 5 minutes.
  5. Add 800 l of LB Broth to both the tubes. Incubate the tubes for 1 hour at 37o C to allow the bacteria to recover and to express the antibiotic resistance marker encoded by the plasmid.
  6. Take four LB agar plates containing ampicillin, X-Gal, IPTG and label them as control, A, B and C. Plate 200 l of culture from the ‘control’ tube and plate it on the corresponding plate with a sterile spreader. Plate 50 l, 100 l and 200 l of cell cultures from the 'transformed' tube on the plates labeled as A, B and C, respectively.
  7. Store at room temperature till the plates are dry.
  8. Incubate the plates overnight at 37 oC
TRANSFORMATION OF COMPETENT CELLS[5]
  1. Kanamycin / Chloramphenicol( based on the plasmid)
  2. 450 ml distilled water
  3. Agar LB broth

Equiments

  1. Cooling Centrifuge
  2. Micropipettes and Sterile tips
  3. Orbital Shaker/Incubator
  4. Standard Glasswares (Conical flasks, measuring cylinder)
  5. Distilled water
  6. Ice bath
  7. Spectrophotometer/colorimeter

Procedure

  1. Among the 2 Competent cell tubes label one as test sample (Tube1), add 5ul of the given plasmid DNA to it, mix well by taping the tube and to the other tube do not add any DNA to maintain as negative control (Tube2).
  2. Incubate the tubes in ice for 30 minutes.
  3. Give heat shock by transferring the microfuge tubes to a 42°C water bath for not more than 90 seconds.
  4. Place the microfuge tubes in ice for 10 minutes. Add 400ul of sterile LB broth medium to them.
  5. Incubate the microfuge tubes at 37°C with agitation for 45 minutes.
  6. Use L rod evenly spread 100 pl of cells from each tube on LB agar plates 1 and 2 (Plate one for Tube 1 and Plate 2 for Tube 2).
  7. Incubate the plates at 37°C for 18 hours and examine the results.

Transformation

  1. Take 200 l of the above cell suspension in two 2.0 ml collection tubes and label them as ‘control’ and ‘transformed’. Add 2 l of plasmid DNA to the tube labeled as transformed and mix well.
  2. Incubate both the tubes on ice for 30 minutes
  3. Transfer them to a preheated water bath set at a temperature of 42oC for 2 minutes (heat shock).
  4. Rapidly transfer the tubes on ice-bath. Allow the cells to chill for 5 minutes.
  5. Add 800 l of LB Broth to both the tubes. Incubate the tubes for 1 hour at 37o C to allow the bacteria to recover and to express the antibiotic resistance marker encoded by the plasmid.
  6. Take four LB agar plates containing ampicillin, X-Gal, IPTG and label them as control, A, B and C. Plate 200 l of culture from the ‘control’ tube and plate it on the corresponding plate with a sterile spreader. Plate 50 l, 100 l and 200 l of cell cultures from the ‘transformed’ tube on the plates labeled as A, B and C, respectively.
  7. Store at room temperature till the plates are dry.
  8. Incubate the plates overnight at 37oC
MINIMAL MEDIA (M9) MEDIUM XYLOSE PREPARATION TO FAVOR THE GROWTH OF RECOMBINANT E coli [10]
  1. Na2HPO 4·7H2O
  2. NH4Cl, M9 salt solution
  3. NaCl, 20% xylose
  4. 1 M CaCl2
  5. 1 M MgSO4
  6. KH2PO4

Procedure

  1. M9 Saline (10X): 100 mL of M9 Saline is required. When diluted to working concentrations, the final concentrations are 33.7 millimolar (mM) Na2HPO4, 22.0 mM KH2PO4, 9.35 mM NH4Cl, and 8.55 mM NaCl. 20% Xylose: Add 20 ml of 20% xylose solution for a final concentration of 0.4%.
  2. 1 M MgSO4: Add 1 mL of 1 M MgSO4 solution to give a final concentration of 1 millimolar (mM) MgSO4.
  3. 1 M CaCl2: Add 0.3 mL of 1 M CaCl2 solution to give a final concentration of 0.3 millimolar (mM) CaCl2.
DETERMINATION OF DRY CELL WEIGHT[7]
  1. LB media
  2. Xylose
  3. Chloramphenicol
  4. Conical flask

Equiments

  1. Colorimeter
  2. Weighing balance

Procedure

  1. Incubate the bacteria overnight in 4 ml of LB medium containing the appropriate selective antibiotic kanamycin (pSB3K3).
  2. Transfer the bacteria from the preculture to a 20 mL flask containing M9 medium containing xylose.
  3. Grow the bacteria for 24 h at an incubation temperature of 37 °C with constant agitation at 177 rpm.
  4. After the incubation period, measure the optical density of the bacterial culture using 600 nm wavelength light and divide the culture to achieve optical density values ​​of 0.25, 0.5, 0.75, and 1 in separate 4 mL bacterial volumes.
  5. Dilute Weigh the empty Eppendorf tube and record its initial weight. Carefully transfer 4 mL of bacterial culture to an Eppendorf tube and centrifuge the contents at 16,000 x g for 3 min.
  6. After centrifugation, place the Eppendorf tube with the lid open in a hot oven at 60 °C for 12 h.
  7. After 12 hours of incubation, measure the net weight of the Eppendorf tube containing the bacterial pellet.
PRK TOXICITY TEST TO DETERMINE THE RELATIONSHIP BETWEEN EXPRESSION OF GENE AND CELL GROWTH
  1. LB media
  2. Antibiotic
  3. M9 xylose

EQUIPMENTS :

  1. Colorimetry

Procedure:

  • Pre-culture the bacteria overnight in a 4 ml LB medium containing antibiotics.
  • Transfer the pre cultured bacteria to a separate tube containing 4 mL of M9 medium containing xylose.
  • Place the test tube in a standard incubator and incubate the bacteria for 12 hours with continuous agitation at 177 rpm.
  • Measure the optical density (OD) of the bacterial samples using 600 nm light at the beginning (0 h) and end of the 12 h incubation.
  • Be sure to perform these measurements three times.
TOTAL SOLUTION TO CONFIRM THE PRESENCE OF ENZYME[15]
  1. LB media
  2. Antibiotic
  3. M9 xylose
  4. 4% xylose

Equiments

  1. Centrifuge
  2. Colorimeter

Procedure:

  1. Pre-incubate the bacterial culture overnight by transferring a single colony to 4 ml of LB medium containing antibiotics. To reach logarithmic growth phase (approximately 3 hours), expand the bacterial culture to 30 ml of LB medium by adding 1/100 of the preincubated culture and an appropriate selective antibiotic.
  2. Once the desired growth phase is reached, centrifuge the bacterial culture at 22 °C and 3200 rpm for 5 min.
  3. Next, change the medium to His M9 medium supplemented with 4% xylose and 0.1% LB.
  4. Continue culturing the bacteria for 24 h in an incubator that maintains both O2 and 5% CO2 levels.
  5. During this 24-hour incubation period, measure the optical density (O.D.) of the culture every 6 hours.
RUBISCO FUNCTIONALITY TEST[8]
  1. LB medium
  2. Antibiotic
  3. Rhamnose
  4. 20 mM sodium phosphate
  5. 500 mM NaCl buffer
  6. 0.2 mg/ml lysozyme
  7. 20 µg/ml DNAse
  8. 1 mM MgCl2
  9. 1 mM PMSF
  10. 750 µL bicine buffer
  11. 2 M MgCl2
  12. 450 µL H2O
  13. 250 mM NaHCO3
  14. 12.5 mM ribulose-1,5-bisphosphate solution

Equiments

  1. Centrifuge
  2. HPLC
  3. Colorimeter

Procedure:

  1. Transformed cells are cultured overnight in LB medium containing specific antibiotics. Incubate the culture at 37°C.
  2. Dilute the overnight culture 1:50 in LB medium containing antibiotics to allow cells to continue growing at 37 °C.
  3. Once the culture reaches an optical density at 600 nm (OD600) of 0.6-0.8, reduce the temperature to 20 °C.
  4. Trigger protein expression under the control of the T7 promoter by adding rhamnose to a final concentration of 0.1%.
  5. To harvest cells, centrifuge the culture at 4°C.
  6. Remove and discard the supernatant. For each gram of cell pellet, add 5 ml of buffer containing 20 mM sodium phosphate and 500 mM NaCl. Add 0.2 mg/ml lysozyme, 20 μg/ml DNAse, 1 mM MgCl2 and 1 mM PMSF to this mixture. The mixture is stirred for 30 minutes at 4°C. Centrifuge the mixture for 30 min at 4°C and transfer the resulting supernatant (cell extract) to a new tube.
  7. For the RuBisCo activity test, add NaHCO3, ribulose 1,5-diphosphate solution, and cell extract as the last ingredients.
  8. Run the reaction at 37 °C with continuous purge of carbon dioxide.
  9. Samples were taken at 5 minute intervals and immediately frozen at -20°C. Before analyzing the samples by HPLC, a preparatory step is centrifugation through an Amicon Ultra filter device with a 10 kDa cutoff.
    10. Finally, the prepared samples are analyzed by HPLC according to established HPLC protocols.

ISOBUTANOL PRODUCTION

ISOBUTANOL QUANTIFICATION
  1. Ethyl acetate
  2. Isobutanol

EQUIPMENTS

  1. Gas chromatography apparatus
  2. Colorimeter
  3. Mass spectrometer

PROTOCOL:

  1. Isobutanol extraction and quantification:
    1. Harvest the cells expressing the pathway and pellet them by centrifugation.
    2. Resuspend the cell pellet in an appropriate solvent, such as ethyl acetate, which can efficiently extract isobutanol from the aqueous phase.
    3. Incubate the cell suspension with the solvent, shaking or vortexing to facilitate extraction.
    4. Separate the solvent phase (containing isobutanol) from the aqueous phase(gas stripping / liq-liq extraction.
    5. Analyze the extracted solvent phase using gas chromatography (GC) or liquid chromatography (LC) coupled with a suitable detector, such as a flame ionization detector (FID) or mass spectrometry (MS).
    6. Quantify the amount of isobutanol in the extracted sample by comparing its peak area or peak height to a standard curve generated using known concentrations of isobutanol.
  2. Optical density and growth rate measurements:
    1. Monitor the growth of the bacterial culture over time by measuring the optical density at a specific wavelength (e.g., OD600) using a spectrophotometer.
    2. Take periodic OD measurements at regular intervals (e.g., every hour) to assess the growth rate of the culture. Compare the growth rate and final OD between the isobutanol-producing strain and control strains lacking the pathway or expressing only a subset of the genes. A slower growth rate or lower final OD in the isobutanol-producing strain may indicate a metabolic burden due to isobutanol production.
  3. Isobutanol standard curve:
    1. Prepare a series of dilutions with known concentrations of isobutanol, ranging from low to high concentrations.
    2. Analyze these dilutions using the same method described in Step 1e (GC, LC, or other suitable technique).
    3. Generate a standard curve by plotting the known concentrations of isobutanol against their corresponding peak areas or heights.
    4. Use this standard curve to quantify the concentration of isobutanol in your experimental samples.

GROWTH OF CYANOBACTERIA [13]

CYANOBACTERIA GROWTH
  1. BG-11 media

EQUIPMENTS

  1. Falcon tubes
  2. Ethanol
  3. Erlenmeyer flask
  4. Cotton stoppers
  5. Aluminum caps

PROTOCOL:

  1. Autoclave the Erlenmeyer flask with the cotton stopper and aluminum cap (P1) (switch on the autoclave 3 hours before use).
  2. Follow the instructions for the hood.
  3. eat the BG-11 medium to 25°C (further heat to a temperature of 30°C).
  4. Bring the Falcon tube and autoclave flask to the vent (spray everything with ethanol).
  5. Using a pipette, transfer the desired amount of her BG-11 to each flask.
  6. Transfer the required amount of cyanobacteria to the flask (mix with a pipette).
  7. Please cap the bottle to avoid causing cross-contamination.
  8. Place the flask in a shaker/incubator (starting at 25 °C and increasing it to 30 °C).
  9. Place a light inside the incubator.
SPREAD PLATE OF CYANOBACTERIA
  1. Antibiotics
  2. Growth media

Equiments

  1. Bench and hood
  2. Tungsten spreader
  3. Pipette Plates

PROTOCOL

  1. Spray the seats and hood.
  2. Place the pipette and plate in the hood and spray.
  3. Set up the Bunsen burner.
  4. Light the gas and use the igniter to light the fire.
  5. Add 4 mL of growth medium and antibiotics to a 15 mL culture tube.
  6. Place the tungsten shaker in the flame of a Bunsen burner and sterilize it until it turns bright orange.
  7. Wait for the spreader to cool and test it by pressing the end into the agar.
  8. If no sizzling sound is observed, this indicates that the object has cooled down.
  9. Pick up colonies with a chilled spreader and place into tubes.
  10. Rotate the spreader to ensure the entire colony is protected.

ANALYTICAL

GAS CHROMATOGRAPHY MASS SPECTROMETRY
  1. GC-MS instrument
  2. Sample preparation equipment
  3. GC columns
  4. Carrier gas
  5. Transfer line
  6. Ionization source
  7. Mass analyzer
  8. Detector
  9. Data system
  10. Solvents
  11. Calibration standards
  12. Reference spectra

PROTOCOL

  1. Prepare the sample. The sample should be prepared by extracting the compounds of interest from the matrix. This can be done using a variety of methods, such as solvent extraction, solid-phase extraction, or headspace sampling.
  2. Inject the sample into the GC. The sample is injected into the GC oven using a syringe or an autosampler.
  3. Choose the appropriate column and carrier gas. The type of column and carrier gas will depend on the compounds that you are analyzing.
  4. Set the oven temperature program. The oven temperature program is a series of temperatures that the oven will go through during the analysis. This program is designed to separate the compounds based on their boiling points.
  5. Transfer the separated compounds to the MS. The separated compounds are transferred to the MS using a transfer line.
  6. Ionize the compounds. The compounds are ionized in the MS using an ionization source.
  7. Separate the ions by mass-to-charge ratio (m/z). The ions are separated by mass-to-charge ratio in the MS analyzer.
  8. Detect the ions. The ions are detected at the end of the analyzer and their m/z values are measured.
  9. Identify the compounds. The compounds are identified by comparing their m/z values to a library of known compounds.

MASS SPECTROMETRY

  1. Prepare the sample. The sample should be prepared by dissolving it in a solvent. The solvent should be compatible with the ionization source.
  2. Introduce the sample into the MS. The sample can be introduced into the MS using a variety of methods, such as direct injection, electrospray ionization (ESI), or matrix-assisted laser desorption ionization (MALDI).
  3. Ionize the sample. The sample is ionized in the MS using an ionization source. The ionization source creates ions by removing electrons from or adding protons to the molecules in the sample.
  4. Separate the ions by mass-to-charge ratio. The ions are separated by mass-to-charge ratio in the MS analyzer. There are a variety of different mass analyzers available, such as quadrupole mass analyzers, time-of-flight mass analyzers, and ion trap mass analyzers.
  5. Detect the ions. The ions are detected at the end of the analyzer and their m/z values are measured.
  6. Identify and quantify the compounds. The compounds are identified by comparing their m/z values to a library of known compounds. The compounds can be quantified by measuring the intensity of the ions at their characteristic m/z values.
HPLC FOR PURIFICATION OF PRODUCT
  1. Crude product sample
  2. HPLC system
  3. HPLC column
  4. Mobile phase
  5. Fraction collector
  6. UV-Vis spectrometer
  7. Ultrafiltration unit
  8. Lyophilizer 0.45 µm filter
  9. Syringe or pump
  10. Gradation mixer
  11. Fraction collector tubes
  12. Gel filtration media

PROTOCOL

  1. Prepare the crude product sample. The crude product sample should be filtered through a 0.45 µm filter to remove any particulate matter.
  2. Choose the appropriate HPLC column and mobile phase. The type of HPLC column and mobile phase will depend on the properties of the product you are purifying. For example, if you are purifying a protein, you may use a reversed-phase HPLC column with a mobile phase consisting of water and acetonitrile.
  3. Load the crude product sample onto the HPLC column. This can be done using a syringe or pump.
  4. Apply a gradient elution program. A gradient elution program gradually changes the composition of the mobile phase over time. This allows for the separation of the product from impurities.
  5. Collect the eluted fractions. The eluted fractions will contain the product and impurities.The fractions containing the product of interest should be collected.
  6. Analyze the eluted fractions. The eluted fractions can be analyzed using a variety of methods, such as UV-Vis spectroscopy, mass spectrometry, or fluorescence spectroscopy.This analysis will help to identify the fraction containing the product of interest.
  7. Pool the fractions containing the product of interest. The pooled fractions can then be concentrated and dried to obtain the purified product.

[1] Selective and Sensitive Method for PCR Amplification of Escherichia coli 16S rRNA Genes in Soil. G. Sabat, P. Rose, W. J. Hickey, J. M. Harkin

[2] Analysis of Endonuclease R·EcoRI Fragments of DNA from Lambdoid Bacteriophages and Other Viruses by Agarose-Gel Electrophoresis Robert B. Helling, Howard M. Goodman, Herbert W. Boyer.

[3] Identification of bacteria in a biodegraded wall painting by denaturing gradient gel electrophoresis of PCR-amplified gene fragments coding for 16S rRNA. S Rölleke, G Muyzer, C Wawer, G Wanner, W Lubitz.

[4] The antimicrobial mechanism of electrochemically activated water against Pseudomonas aeruginosa and Escherichia coli as determined by SDS‐PAGE analysis Get access Arrow. T.E. Cloete, M.S. Thantsha, M.R. Maluleke, R. Kirkpatrick.

[5] Studies on transformation of Escherichia coli with plasmids. Douglas Hanahan.

[6] Engineering carbon fixation in E. coli: from heterologous RuBisCO expression to the Calvin–Benson–Bassham cycle. Niv Antonovsky, Shmuel Gleizer, Ron Milo.

[7] Determination of biomass dry weight of marine microalgae. C. J. Zhu & Y. K. Lee.

[8] Directed evolution of RuBisCO hypermorphs through genetic selection in engineered E.coli. Monal R. Parikh, Dina N. Greene, Kristen K. Woods, Ichiro Matsumura.

[9] Protein Engineering, Design and Selection, Volume 19, Issue 3, March 2006, Pages 113–119.

[10] Enhancing the protein production levels in Escherichia coli with a strong promoter. Hanna Tegel, Jenny Ottosson, Sophia Hober. First published: 14 December 2010.

[11] Enhancing E. coli isobutanol tolerance through engineering its global transcription factor cAMP receptor protein (CRP). Huiqing Chong, Hefang Geng, Hongfang Zhang, Hao Song, Lei Huang, Rongrong Jiang. First published: 12 October 2013.

[12] Assembly of BioBrick Standard Biological Parts Using Three Antibiotic Assembly. Reshma Shetty, Meagan Lizarazo, Randy Rettberg, Thomas F. Knight

[13] Transformation in Cyanobacteria. Ronald D. Porter. Pages 111-132 | Published online: 25 Sep 2008.

[14] Chapter 2 - Isolation and Purification of Industrial Enzymes: Advances in Enzyme Technology. Soumya Mukherjee.

[15] Compound identification in GC-MS by simultaneously evaluating mass spectrum and retention index. Xiaoli Wei, Imhoi Koo, Seongho Kim, and Xiang Zhangcorresponding.