Experiments

Overview

Our wetlab consisted of two parts: experiments using Azospirillum brasilense sp7 and E. coli. Azospirillum brasilense is our chassis organism but since we are the first iGEM team to work with it, we also decided to simultaneously work with E. coli as a model system. We decided to work in Prof. Anil Kumar Tripathi’s lab at Banaras Hindu University as he is one of the leading scientists working on Azospirillum.

To begin with we aimed to characterize the activity of the alcohol inducible promoter, known as the exaA promoter in hypoxic environments. In order to do so we planned on performing a pellicle experiment assay which would enable Azospirillum to migrate to a microaerobic environment which would have allowed us to ascertain the activity of the exaA promoter. We also planned on working with the fdhF promoter, which is a well characterized hypoxia promoter in E. coli. To do so we planned on cloning the fdhF promoter upstream of the lacZ gene in the pcZ backbone by traditional cloning.

As a proof of concept we planned on cloning the acdS gene downstream of the exaA promoter in the pCZ vector and mobilizing this engineered plasmid into A. brasilense by biparental mating. As a qualitative assay we wanted to monitor the growth curves of the transconjugated Azospirillum in order to determine the metabolic load/benefit of our engineered plasmid.

In order to have a proper control to check hypoxic expression vs regular expression we also planned on cloning the acdS gene downstream to a constitutive promoter in the pAK002 plasmid and use this as a comparative study for all of our experiments involving acdS expression.

To characterize the activity of the ACC deaminase gene we planned on performing a spectrophotometric assay to detect the product formed as a result of the enzyme catalyzed reaction.

As we were working with two organisms we decided to conduct a cross-species promoter study in order to characterize a new probable alcohol inducible/hypoxia promoter in E. coli. To characterise exaA in E. coli we planned on cloning it upstream of the mCherry reporter gene by using RF cloning. Since it might have been difficult to perform the ACC assay in Azospirillum we planned on performing it in E. coli and then customising the protocol for E. coli. To do the same we decided to clone the acdS gene downstream of T7 overexpression promoter.

To find the detailed protocol for all these procedures refer the following tabs.

← Engineering

RF Cloning

  1. First step of RF cloning involves designing primers for the primary PCR. Primers are made by taking two parts into consideration :
    1) The sequence flanking the region where target gene has to be introduced in the recipient vector
    2) 5’ and 3‘ ends of the target gene.
  2. Primary PCR : This PCR product, called the megaprimer, will have the target gene flanked by regions complementary to the regions flanking the site of insertion in the target vector. The plasmid with the target gene or linearised target gene will be used as the template of this PCR.
  3. Secondary PCR : Purified PCR product from the primary PCR will be used for the secondary PCR. The template vector will be the recipient vector and during amplification, the target gene will be inserted into the recipient vector.
  4. After the secondary PCR, the product is digested with Dpn 1 to degrade methylated parent vectors. The digested product is introduced in E.coli by electroporation.

Primary PCR protocol :

  1. Initial denaturation: Run for 2 min at 95°C
  2. Repeat 30 times:
    3. a) Denaturation: 30s at 95°C
    b) Annealing: 30s at the desired annealing temperature
    c) Extension: 1 min/1 kb template at 72°C
    Note : The rate of extension depends on the polymerase being used in the PCR mix. The rate for Pfu polymerase has been mentioned.
  3. Final extension: Run for 10 min at 72°C
  4. Run PCR products on an agarose gel and purify it using a PCR purification kit.
  5. Dilute the final concentration to 100ng/μL

    Secondary PCR protocol :
  1. Initial Denaturation: Run for 5 min at 95°C
  2. Repeat 30 times:

    3. a)Denaturation: 30s at 95°C
    b)Annealing: 30s at the desired annealing temperature
    c)Extension: 1 min + 1 min/1 kb template at 72°C
    Note: The rate of extension depends on the polymerase being used in the PCR mix. The rate for Pfu polymerase has been mentioned.
  3. Final extension : Run again for 10 min at 72°C.
  4. Run PCR products on an agarose gel.
  5. Digest the PCR products using DpnI enzyme at 37℃ for 3 hrs.
  6. Transform the product into electrocompetent E. coli by electroporation.

α-ketobutyrate standard curves

  1. Make 2M Tris HCl pH 8.5
  2. Volume to be made - 200mL
  3. Procedure
    4. a) Weigh 48.456g of Tris base and add it to 140mL of milliQ
    b) Adjust pH to 8.5 by adding conc HCl and let it cool to room temp and check pH again - readjust if changed
    c) Make volume to 200mL and autoclave
  4. Make 0.1M Tris HCl pH 8.5
    1. Volume to be made - 100 mL
    2. Procedure
      3. a)Take 5mL 2M Tris HCl and add to 95mL of autoclaved milliQ
  5. Make 100mM stock of α-KB(AKB)
    1. Volume to be made - 25mL, in a falcon
    2. Procedure
      3. a) Measure 0.255g of AKB and add it to 25mL of 0.1M Tris HCl
  6. Make 10mM working solution of AKB
    1. This is to be done just before working only
    2. Volume to be made - 5mL
    3. Procedure
      4. a) Take 500uL of 100mM AKB and add to 4.5mL of 0.1M Tris HCl
  7. Make aliquots of AKB of different concentrations
    1. Volume to be made - each aliquot is finally 200uL
    2. Label them as
      3. a) 0.5mM AKB - add 10uL of 10mM AKB in 190uL of 0.1M Tris HCl
      b) 1.0mM AKB - add 20uL of 10mM AKB in 180uL of 0.1M Tris HCl
      c) 1.5mM AKB - add 30uL of 10mM AKB in 170uL of 0.1M Tris HCl
      d) 2.0mM AKB - add 40uL of 10mM AKB in 160uL of 0.1M Tris HCl
      e) 2.5mM AKB - add 50uL of 10mM AKB in 150uL of 0.1M Tris HCl
      f) 3.0mM AKB - add 60uL of 10mM AKB in 140uL of 0.1M Tris HCl
      g) 3.5mM AKB - add 70uL of 10mM AKB in 130uL of 0.1M Tris HCl
      h) 4.0mM AKB - add 80uL of 10mM AKB in 120uL of 0.1M Tris HCl
      i) 4.5mM AKB - add 90uL of 10mM AKB in 110uL of 0.1M Tris HCl
      j) 5.0mM AKB - add 100uL of 10mM AKB in 100uL of 0.1M Tris HCl

Expression of acdS gene

  1. Transform E.coli BL21DE3 with pHis17-acdS
  2. The next morning, pick up a patch from the plate and inoculate it in 10mL LB + Amp
  3. Incubate it at 37°C, 180rpm until turbidity is observed
  4. Prepare 4 test tubes, labelled as:
    5. a) Uninduced
    b) 0.25mM IPTG
    c) 0.50mM IPTG
    d) 1.00mM IPTG
  5. Add the following to the tubes:
    6. a) 10mL LB+Amp
    b) 100uL of primary culture
  6. Incubate at 37°C, 180rpm until the OD is between 0.4-0.8
  7. Once the OD is in range, add the following to the tubes:
    8. a) Uninduced - 0uL of 1M IPTG
    b) 0.25mM IPTG - 2.3uL of 1M IPTG
    c) 0.5mM IPTG - 4.6uL of 1M IPTG
    d) 1mM IPTG - 9.1uL of 1M IPTG
  8. Induce for 3 hours at 37°C
  9. Take 3mL of each culture, pellet it down, flash freeze it, and store it at -80°C.

    10. ACC deaminase assay

    1. Transform E. coli BL21(DE3) with pHis17-acdS
    2. Do primary inoculation in 10mL LB + Amp media, 37°C for 18 hours
    3. Harvest cells by centrifugation, at 6000g for 10 mins in a 4° incubator
    4. Throw out the supernatant
    5. Wash twice with 0.1M Tris-HCl (pH 7.5) (Resuspend, pellet down, throw supernatant)
    6. For secondary inoculation, do it in duplicate, 10mL M9 minimal media with 0.5mM IPTG (5uL of 1M IPTG), for 18 hours
    7. The cells are then pelleted and resuspended in 0.1M Tris-HCl (pH 8.5)
    8. The cells are then lysed by sonication
    9. In a 1.5mL MCT, 200uL of sonicated cells are added
    10. 20uL of 0.5M ACC is added
    11. The suspension is vortexed, then incubated at 30°C for 15 mins
    12. 1mL of 0.56M HCl is added
    13. The mixture is vortexed, then centrifuged at 25°, 16000g, 5 mins
    14. Into a glass test tube, add 1mL of supernatant and 800uL of 0.56M HCl, vortex
    15. Then 300uL of 0.2% 2,4-DNP in 2M HCl is added to the tube
    16. The contents are vortexed and incubated at 30°C for 30 mins
    17. 2mL of 2N NaOH is added and mixed
    18. The absorbance of the mixture is measured at 540nm


    References:

    [1] Farajzadeh D, Aliasgharzad N, Sokhandan Bashir N, Yakhchali B. Cloning and characterization of a plasmid encoded ACC deaminase from an indigenous Pseudomonas fluorescens FY32. Curr Microbiol. 2010 Jul;61(1):37-43. https://doi.org/10.1007/s00284-009-9573-x

    [2] Mamoru Honma & Tokuji Shimomura (1978) Metabolism of 1- Aminocyclopropane-1-carboxylic Acid, Agricultural and Biological Chemistry, 42:10, 1825-1831, https://doi.org/10.1080/00021369.1978.10863261

    [3] Shah S, Li J, Moffatt BA, Glick BR. Isolation and characterization of ACC deaminase genes from two different plant growth-promoting rhizobacteria. Can J Microbiol. 1998 Sep;44(9):833-43. PMID: 9851025http://dx.doi.org/10.1139/w98-074

    [4] Penrose DM, Glick BR. Methods for isolating and characterizing ACC deaminase-containing plant growth-promoting rhizobacteria. Physiol Plant. 2003 May;118(1):10-15. https://doi.org/10.1034/j.1399-3054.2003.00086.x

Genomic Extraction and Amplification

  1. Harvest cells from 2mL of bacterial culture by centrifuging at 14,000 rpm for 2 mins. Collect the pellet and discard the supernatant.
  2. Resuspend cells in 567uL of TE buffer.
  3. Add 30uL of 10% SDS and 3uL of proteinase K to the solution and invert mix it.
  4. Incubate at 37°C for 40-60 mins till the solution is colorless.
  5. Add 100uL of 5M NaCl, mix by vortexing and add 80uL of pre-warm CTAB solution.
  6. Mix it by vortexing and incubate at 65°C for 10 mins in a water bath.
  7. Add equal volume of PCI (Phenyl Chloroform Isoamyl alcohol). Mix it by vortexing for about 2 mins.
  8. Centrifuge at 14,000 rpm for 10 mins. Separate the upper aqueous layer in another eppendorf (take around 600uL) and add 0.6 times volume (usually 360uL) of chilled isopropanol.
  9. Centrifuge at 14,000 rpm for 10 mins at room temperature.
  10. Discard supernatant and wash pellets with 1mL of 70% ethanol.
  11. Add 0.7mL of 100% ethanol to 0.3mL of water in a 1.5mL eppendorf tube.
  12. Centrifuge at 14,000 rpm for 5 mins at room temperature.
  13. Discard supernatant and air dry the pellet in 37°C for 5 mins.
  14. Add 50uL of NFW, vortex properly and add 1uL of RNAse.
  15. Incubate at 37°C for 1 hour. Store it in 4°C if required.

Azospirillum specific PCRs

The following modified master mix is used for PCR involving the Azospirillum genome (for both A. brasilense and A. lipoferum).

  1. Master Mix composition:
    2. a) Taq polymerase - 1uL
    b) Taq buffer (10X)- 10uL
    c) Primers (forward and reverse)- 5uL each
    d) dNTP - 5uL
    e) DMSO - 5uL
    f) NFW- add so that the total volume is 100uL
    g) Template- 0.5uL for genomic DNA and 2uL of plasmid DNA
  2. After adding all these components in a 1.5mL eppendorf tube, pipette mix properly and distribute the master-mix in PCR tubes kept in ice.
  3. Short spin the PCR tubes and set the tubes in PCR machine. DMSO is added in the PCR master-mix because Azospirillum genome is GC rich.

PCR cycle:
a) Initial denaturation: 95°C for 5 mins
b) Denaturation: 95°C for 1 min
c) Annealing: Optimized temperature determined by gradient PCR for 1 minute
d) Extension: 72°C for 1 min (for 1 kb gene or less), 2 min (for 2 kb gene) and 3 min (for 3 kb gene)
e) Storage: 4°C for as long as required

Traditional Cloning

  1. Restriction digestion:
    For a 50uL double digestion, add the below constituents in the following order:
    a) NFW - 23.5uL
    b) Plasmid - 20uL
    c) Buffer - 5uL
    d) Enzyme 1 - 0.75uL
    e) Enzyme 2 - 0.75uL
    This is followed by overnight incubation at the appropriate temperature depending on the restriction enzyme used.
  2. Antarctic phosphatase reaction: Add the below constituents in the following order:
    a) Digested plasmid- 50uL
    b) NFW- 24.5uL
    c) Antarctic phosphatase buffer 10x - 5uL
    d) Antarctic phosphatase enzyme- 0.5uL
    Total- 50uL
    The reaction is setup in a thermocycle with a 37°C step for 30 mins, followed by 80°C for 2 minutes to inactivate the enzyme.
  3. Ligation of an insert in the vector of interest: Add the below constituents in the following order into a 1.5mL Eppendorf:
    a) NFW - 5uL
    b) Digested plasmid - 8uL
    c) Insert - 4uL
    d) Buffer - 2uL
    e) Enzyme - 1uL
    f) ATP - 0.5 uL
    Place the Eppendorf in a PCR machine for 10 minutes at 22°C, followed by 3 hours at 16°C.
  4. The ligation product is then transformed into E. coli DH5α and plated on the appropriate antibiotic media.
  5. Colony PCR:
    Preparation of 1mL Titron X:
    a) Add 2-3uL of 100x Titron X to 1mL of autoclaved ddH2O.
    b) Add 20uL of this mix in the respective 1.5mL Eppendorf tubes.
    c) Pick up individual colonies from the plate and mix it properly with titron X and then short vortex the tubes.
    d) Insert the Eppendorf tubes in boiling water for 10 mins and short vortex again.
    e) Centrifuge at 14000 rpm for 5 mins. This is now the template for the PCR reaction.
    f) Template should be added in the master mix such that template is 0.1 times the volume of master mix.

Competent cell preparation

  1. Setup a primary 5mL primary culture and allow overnight incubation.
  2. Inoculate secondary culture from primary culture. For E. coli, 0.5% - 1% volume from the primary culture should be inoculated.
  3. Harvest 50mL of cells when the OD600 of the culture is around 0.6.
  4. Centrifuge the culture at 4000rpm at 4°C for 10 mins.
  5. Discard the supernatant and resuspend the cells in chilled 10mL 100mM CaCl2 and incubate it in ice for 30 mins.
  6. Centrifuge at 4000rpm for 10mins at 4°C. Discard the supernatant.
  7. Dissolve the cells in 25% (250mL) glycerol and 75% (750mL) 100 mM CaCl2.
  8. Pour it in 1.5mL eppendorf tubes and store it in -80°C for further use.

Conjugation: Biparental Mating

  1. Plating the donor and the recipient together:
    2. a) Day 1: Primary inoculation of E. coli S17.1 and Azospirillum brasilense Sp7 in LB with antibiotics.
    b) Day 2: Secondary culture of S17.1 (50-100uL in 5 mL LB) and Sp7 (250uL in 5 mL LB) with appropriate antibiotics.
    c) Monitor OD till it reaches 0.6 - 0.8.
    d) Collect 1:3 ratio of E. coli S17.1: Azospirillum brasilense and mix.
    e) Centrifuge at 4000 rpm for 3 mins.
    f) Discard the supernatant and resuspend in 1mL of 0.85% saline solution.
    g) Add 100uL of above mixture at the center of a blank LBA plate dropwise.
    9) Keep the plate without parafilm in a humid chamber (made by layering tissue papers and cotton inside a tip box and wetting it) at 30°C.
    10) Check 2-3 days later for growth and then move onto the spreading step.

  2. Spreading and dilution:
    3. a) Take 1mL of 0.85% saline solution in 1.5mL eppendorf.
    b) Using a 1mL tip, pickup the central portion of Azospirillum brasilense Sp7 growth. It is observed that there is growth of Azospirillum in the center and surrounding its periphery, with a distinct margin, is E. coli growth.
    c) Swirl and add to the 1mL saline solution in the eppendorf. Keep on scraping and swirling and mixing till nearly all Azospirillum has been removed from tip. Make sure not to touch E. coli.
    d) Pipette mix the saline solution and Azospirillum together using 1 mL tip. As the mixture will have some solid suspended, allow it to sediment by keeping it vertical for some time.
    e) Meanwhile, take 4 eppendorf tubes for dilution and label them (-1,-2,-3 and -4).
    f) Make 5 plates having Ampicillin (for Azospirillum) and the corresponding antibiotic of the plasmid to be mobilized. Label the 5 plates as Sp7:plasmid from 0 to -4.
    g) Add 900uL saline solution to the -1 tube and then add 100uL of supernatant (having Azospirillum) formed from the previous tube. Continue doing the same for the subsequent tubes (serial dilution).
    h) For spreading, take 100uL from each medium and drop it at various locations on separate plates and spread thoroughly.

Pellicle

    Step 1: Preparing primary culture
  1. Take 5mL of complete minimal malate solution (NFB+) in test tubes.
  2. Add appropriate concentrations of antibiotics.
  3. Scrape out single colonies from the respective Azospirillum plate using a pipette.
  4. Discard the pipette tip inside the test tubes.
  5. Keep the test tubes in an incubator at 30°C.
  6. Wait until the OD becomes 1.
    Step 2: Preparing secondary culture
  1. After the OD becomes 1, inoculate secondary culture.
  2. Add 5mL of minimal malate media in a test tube and with the appropriate antibiotics.
  3. Add 200uL of primary culture to this test tube.
  4. Keep it in an incubator for around 8 hours.
    Step 3: NFB- broth preparation
    Add the following solutions in a 10mL falcon:
  1. 500uL of phosphate solution
  2. 500uL of malate solution
  3. 500uL of N2 free salt solution
  4. Add autoclaved ddH2O until the volume is 10mL.
    Step 4: Inoculum preparation
  1. The secondary culture would have an OD of 0.8-0.9.
  2. Collect 1 OD cells from the secondary culture.
  3. Volume that is supposed to be collected = Desired OD/Actual OD.
  4. Centrifuge the cells at 4000 rpm for 3 mins.
  5. Discard the supernatant and resuspend the pellet in 1mL NFB- broth by tapping.
    Step 5: 0.2% Agar (semi-solid media) preparation
  1. Dissolve 0.2g Agar in 85mL ddH2O and autoclave it.
  2. Add 5mL of each solution: phosphate, N2 free salt and malate solutions.
  3. To 30mL solution of this, add 30uL of ampicillin.
    4. Note: Azospirillum brasilense has ampicillin resistant beta lactamase genes in its genome.
    Step 6: Inoculum setup
  1. In 5mL of semisolid media that has still not solidified add the required antibiotics (appropriate with respect to the plasmid inserted in the chassis) and relevant reagents in appropriate amounts.
  2. Pipette 50uL of cell mixture and very slowly and stably release it in the layer just below the top.
  3. Once inoculated, make sure that the test tube undergoes minimum shaking or movement and keep it at 30°C (for growth) or room temperature (if only movement has to be observed) in an extremely stable place.
  4. Check pellicle after 24-48 hours.

EPS Quantification

  1. Centrifuge biofilm samples (15mL of A. brasilense culture) at 15,000g for 20 min.
  2. Resuspend pellets in 30mL of cold 0.2M sulphuric acid solution.
  3. Break biofilm matric with a glass hand homogeniser tube and pestle.
  4. Stir cell suspension at 4°C for 3 hours before centrifuging at 15,000g for 20 min.
    5. Note: this is now known as “EPS solution”
  5. Chemical composition analysis:
    6. a. Precipitate EPS by adding 3 volumes of 100% cold ethanol and incubate on ice for 2 hours.
    b. Precipitate is centrifuged at 17,500g for 2 minutes at 4°C and dried at 50°C overnight.
    c. Perform FTiR spectroscopy.
  6. Total carbohydrate content:
    7. a. 50µL of EPS solution is mixed with 125µL of concentrated sulfuric acid.
    b. 25µL of 10% phenol is mixed in and mixtures is incubated at 95°C water bath for 5 minutes.
    c. Allow mixture to cool then transfer to a 96-well plate.
    d. Measure absorbance at 490nm with a spectrophotometric plate reader.

Measurement →
Access Wiki Tools View Source Code