Team:Exeter/Experiments

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Experiments

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Investigation of B. subtilis Promoters

The aim of this experiment was to test the success rate of multiple B. subtilis promoters. This was done by cloning the 30 different selected Bacillus promoter sequences onto chromoprotein sequences and then light absorbance at the relevant wavelengths was used as indication of successful translation and therefore successful implementation and operation of promoter.

These promoters need to be tested in a B. subtilis environment as this offers a controlled environment of great similarity to the organism in which these promoters would be used in future. This was achieved by carrying the said investigation in a cell free B. subtilis expression plataform. The promoters were all cloned upstream of the chromoprotein code, made by deriving B. subtilis codes with 250bp downstream of every coding sequence being taken (those who overlapped witch other CDS or contained transcription factor binding sites were discarded). Out of the 100bp sequences sp, 30 were randomly selected. If they appear downstream there is a high chance they contained active promoter sequences.

Chromoproteins have a pigmented prosthetic group, which allows the protein, when expressed, to present coloration. By attaching our chosen promoters to these polypeptides, we can use the observation of colour to see where translation was successful and subsequently indicate that the promoter has worked. We chose to test each promoter twice, meaning two chromoproteins were chosen and attached to the 30 promoters. This resulted in 60 samples, along with two negative control tests (one for each protein).

The 62 prepared samples were plated in a well-plate and their absorbance read in 3 wavelengths; 405nm, 450nm and 630nm. These were chosen as they were close to the wavelengths of red and blue light since those are the expected colours of the pigmented proteins.

A visual representation of the well-plate diagraming, which promoters were chosen and which chromoproteins were used can be seen below.

Figure 1: Table showing the arrangement of the 60 DNA constructs and the 2 controls run in the well plate. Red and Blue used to symbolise the pigment expected from those runs, highlighted in respect to which chromoprotein code was attached.

Method/Protocol

The 30 putative promoters had already been cloned downstream of the code for amilCP and meffCP (CP= chromoprotein). This meant the DNA constructs were not included in the below described mix. The 60 resulting constructs were in a clear PCR plate following the arrangement shown in Figure 1.

  1. Amino Acid mix prepared following the JOVE protocol, linked here.
  2. Cell Free master mix was made in a “big batch”. The master mix recipe below was followed:
    • 775 µL B. subtilis Cell Free Extract
    • 775 µL Amino Acid Mix
    • 217 µL Energy Mix
    • 217 µL S30A buffer
    • 186 µL PEG8000
  3. 5 µL of the prepared master mix was then added to all 62 designated wells.
  4. 15 µL from each DNA construct was added to the correct well-plate from the cloning plate, this was done as quickly as possible as the reaction starts immediately, and we wanted to ensure all reactions were equal.
  5. Once completed, the plate was placed in a shaking incubator set to 25ºC and 800 rotations per minute.
  6. Absorbance was then recorded using the Abexxo spectrophotometer every hour for 24 hours in the red/purple and blue wavelengths: 405 nm, 450 nm and 630 nm.

Precipitation Experiment:

This experiment was done to not only confirm we had the capability to precipitate calcium carbonate crystals and manipulate which form we obtain (aragonite or calcite) but also to obtain crystals that we could analyse and compare in future with our bacterium precipitated ones.

Calcite and Aragonite are polymorphs of calcium carbonate, meaning they possess the same empirical and chemical formulae but different crystalline structures. Aragonite’s crystal structure is more resistant to stress than calcite – therefore organisms that inhabit high-energy environments tend to prefer skeletons composed of aragonite. However, calcite has a lower rate of dissolution hence being favoured by organisms that live in greater depth habitats (i.e. the Aragonite compensation depth). For this adaptive reason, we find that most coral species present Aragonite skeletons.

Method/Protocol

We used the following method for the precipitation of calcium carbonate polymorphs;

  1. Two base solutions were made;
    1. 200 mL of 1M CaCl2, made by adding 22.2 g of solid CaCl2 anhydrous to 200 mL of purified water.
    2. 200 mL of 0.1M NaCO3, made by adding 1.2 g of solid NaCO3 to 200 mL of purified water.
  2. The base solutions were then mixed in six different ratios to make up twelve reaction preparations of 1650 µL in 2 mL Eppendorf tubes, two of each ratio. The ratios used are described by the table below.

    3. Figure 2: Table showing the ratios, in micro-litre, used to prepare the 1650µL reactions. Each Reaction prep. Was made twice, totaling in 12 Eppendorf tubes.

  3. The other batch of ratio preparations was placed in 70ºC water bath for 6 minutes.
  4. Two more preparations of the 1:10 ratio were made and left at room temperature, one for 18 hours and the other for 6 minutes. These were taken as no temperature change control solutions.
  5. When the reactions reached the intended times, all were removed from water bath at the same time and immediately put under the microscope where all samples were photographed twice, one picture for the pallet and one picture of the supernatant. The pictures were used as an end point.
  6. All samples, after photographed, were put in the freezer to be kept until necessary again.

The carried reactions and resulting photographs can be summarised in the following tables.

E. coli Competent Cells Preparation

The selected bacteria, E. coli, can only take up small amounts of DNA, therefore to be able to successfully transform this species bacteria, it must undergo physical/chemical treatment> These are called “competent cells” and were prepared following the method described below.

Method/Protocol

Firstly, necessary solutions were prepared:

  1. Transformation Buffer, TF-1 (200 mL)
    • 1.48 g (7.4 g/L) of potassium chloride
    • 0.59 g (2.94 g/L) of potassium acetate
    • 0.30 g (1.5 g/L) of calcium chloride dihydrate
    • 1 µL of 10mM ATP
    • 1 µL of 10U/mL BSA1
    • 1 µL 5U/mL DNA T4 Ligase.
    • 30.00 g ≈ 25 mL (150 g/L) of glycerol

    Make up above solution to a final volume of 190 mL, adjust pH to 6.4 with acetic acid; dispense into 2 x 95 mL aliquots, autoclave and allow to cool. Then add 5 mL (per 95 mL) of 1M Manganese Chloride Tetrahydrate filter sterilised stock (19.8g in 100 mL).

  2. TF-2 (200 mL)
    • 1.48 g (0.74 g/L) of potassium chloride
    • 2.20 g (11 g/L) of calcium chloride dihydrate
    • 30.00 g ≈ 25 mL (150 g/L) of glycerol

    Make up above solution to a final volume of 196 mL, dispense into 2 x 98 mL aliquots, autoclave and allow to cool. Then add 2 mL (per 98 mL) of filter sterilised 0.5M MOPS Buffer (10.47 g in 100 mL), pH 6.8 (adjusted with 5M KOH).

After solutions were prepared competent cell preparation steps can be followed;

  1. A few DH5α E. coli cells from glycerol stock were scraped straight from the -80ºC freezer onto a marked space on a LB-agar plate (with no selection antibiotic). Wire was used to streak the plate thrice and the plate was left to grow overnight at 37ºC.
  2. 5 mL of LB was inoculated in a 50 mL Falcon tube with one colony from the overnight culture. This was then incubated overnight at 37ºC, 200 rpm.
  3. 40 mL of LB was then inoculated in a 250 mL conical flask with 0.4 mL of the overnight culture and left to grow at 37ºC, 200 rpm until optical density reading at 600 nm was 0.4-0.5.
  4. Culture was then transferred onto a 50 mL Falcon tube and harvested using centrifugation at 5000 rpm for 10 minutes at 4ºC.
  5. Supernatant removed and pallet drained. Cells were then resuspended in 8 mL of TF-1.
  6. Falcon tube was then placed on ice for 15 minutes and then centrifuged again at 5000 rpm for 10 minutes at 4ºC.
  7. Pellet was thoroughly drained once more, and re suspended in 4 mL of TF-2.
  8. 100 µL aliquots were created in 2 mL Eppendorf tubes and immediately frozen in liquid nitrogen.
  9. Aliquots were labelled and stored at -80ºC in the freezer.

E. coli Plasmid Extraction (mini-prep):

Method/Protocol

Firstly, the buffer was prepared using the GeneJET kit method, described below.

  1. The provided RNase A solution was added to the Resuspension Solution and mixed thoroughly. This was stored at 4ºC.
  2. 20 mL of the Wash Solution (concentrated) was then diluted in 35 mL of 96% Ethanol.
  3. 40 mL of LB was then inoculated in a 250 mL conical flask with 0.4 mL of the overnight culture and left to grow at 37ºC, 200 rpm until optical density reading at 600 nm was 0.4-0.5.
  4. Both the Lysis and the Neutralization Solution were checked for precipitation – none was found.

We then moved on to the culture growth. The following steps were followed twice, once with Escherichia coli and once with Bacillus subtilis:

  1. Single culture from competent cell culture dissolved in 20 µL of the GeneJET Elution Buffer (from the GeneJET Plasmid Miniprep Kit) and placed in a -20ºC freezer until it was required once more.
  2. Two Escherichia coli cultures were then made, using a liquid medium and full aseptic technique. Two single colonies from a streak plate was chosen and used to inoculate two different mediums, nutrient liquid media (+nb) and Luria liquid medium (+lb). Both mediums were supplemented with as ampicillin a selection antibiotic.
  3. A negative control without the antibiotic within the medium was made.
  4. The 3 prepared cultures were then placed overnight in the shaker incubator at 200rpm and 37ºC.
  5. All three bacterial cultures were then harvested. This was done by centrifuging the cultures at 8000 rpm for 2 minutes at room temperature. The supernatant was decanted, and remainder medium removed. The pellets were then used for plasmid extraction.

One Pot Cloning (Gibson Assembly):

This technique was used to clone and amplify the desired DNA constructs onto the appropriate plasmids. There were 12 different constructs to be tested (all possible combinations of promoter + RBS + CDS + terminator for each bacteria) in E. coli and 18 for B. subtilis. Each one provided as entry vectors, the addition of the appropriate destination vector, type II restriction enzyme (Bsal) and T4 DNA ligase which allows for repeat digestion=ligations cycles to amplify the desired product since the Bsal site is removed from final product. The used destination vector for all set up reactions was px1834.

Method/Protocol

  1. In each PCR tube (one for each reaction) a master mix was set up, composed of;
    • 1 µL of 20 fmol/µL entry vector
    • 1 µL of 20 fmol/µL destination vector
    • 2 µL of 10x Fast Digest Buffer
    • 1 µL of 10mM ATP
    • 1 µL of 10U/mL BSA1
    • 1 µL 5U/mL DNA T4 Ligase.
    • 13 µL of distilled water (solution made up to 20 µL)
  2. Tubes were then put into thermocycler for the following times in indicated temperatures;
    1. 37ºC for 2 minutes
    2. 22ºC for 3 minutes
    3. Repeat above cycles for 25-50 cycles
    4. 37ºC for 5 minutes
    5. 22ºC for 5 minutes
    6. 65ºC for 10 minutes
    7. 10ºC hold

E. coli Transformations:

Experiments were carried out to transform the extracted plasmids onto E. coli, to allow both growth and replication of the circular DNA constructs and B. subtilis implementation in later experiments. The strain used was E. coli DH5α, which was used as a chassis for molecular cloning in order to maximise the constructs present. Twelve E. coli transformations were made, outlined on the tables on the Design webpage.

Method/Protocol

  1. Thaw, on ice, 100 µL aliquot of E. coli competent cells DH5α per transformation plus an extra as a control. The whole tube was used and not re-frozen.
  2. 3 µL of correct plasmid DNA was added and tubes were shaking by rolling.
  3. All tubes were incubated on ice for 40 minutes.
  4. All tubes were then heat-shocked by being transferred into a 42ºC water bath for 2 minutes, then returned to the ice for 2 minutes.
  5. 700 µL of LB medium were added.
  6. All preparations were then incubated at 37ºC, 220 rpm for one hour.
  7. They were then all centrifuged at 8000 rpm for 5 minutes.
  8. 500 µL of the supernatant of all tubes was removed and pellet resuspended in the remaining liquid.
  9. All solutions were then plated out into 200 µL LB-agar plates with the appropriate antibiotic (ampicillin).
  10. All plates were then left in 37ºC incubator overnight with no shaking.

B. subtilis Competent Cell Preparation and Transformation

Method uses several stationary phase processes triggered by nutritional downshift to develop competence in Bacillus subtilis cells. The protocol was adapted for cuvette use.

Method/Protocol

Firstly, the following solutions were prepared.

  1. 10x Medium Base;
    1. Mix together 10 g Yeast extract, 2 g casamino acids in 900 mL of distilled water.
    2. Autoclave the mix, then add 100 mL of filter-sterilized 50% glucose.
  2. 10x Bacillus salts;
    1. Mix together 420 g of (NH4)2SO, 183 g of K2HPO4•3H2O, 460 g of KH2PO, 10 g of Na + citrate, 2 g of Mg(SO4).
    2. Make up solution volume to 1000 mL with distilled water.
  3. Medium A;
    1. Mix together 81 mL of sterile water, 10 mL of 10xMedium A base and 9 mL of 10xBacillus salts.
  4. Medium B;
    1. Mix together 10 mL of Medium A, 0.1 mL of 50mM CaCl2•2H2O and 0.1 mL of 250 nM MgCl2•6H2O.

After solution preparation, cultures could start being made.

  1. Recipient strain was streaked onto half of a Tryptose Blood Agar Base plate. Incubate plate overnight at 37ºC.
  2. A few colonies were inoculated into 9 mL of Medium A in a 10 mL Erlenmeyer flask where contents were mixed thoroughly. Then, 900 µL of mix was pipetted onto a cuvette with an observed optical density of 650 nm from spectrometer
  3. Solution then incubated at 37ºC with vigorous aeration. Optical density at 650 nm was read every 20 minutes, until Optical Density at 650 nm was within 0.1-0.2. A plot showing the OD650 against time was made, only when logarithmic growth ceased (around 2 hours) step 4 was performed.
  4. Transfer 0.05 mL of the culture onto 4.5 mL of pre-warmed Medium B into 19 test tubes – one for each transformation to be performed and one negative control.
  5. Diluted cultures were then incubated at 37ºC with vigorous aeration for 90 minutes.
  6. 1 µg of the designated DNA was added to each tube containing competent cells, and all test tubes were once again incubated at 37ºC with aeration for 30 minutes.
  7. Aliquots were than plated onto selective agar - chloramphenicol was used as a selection antibiotic.

B. subtilis Transformation (alternative protocol)

Method/Protocol

Firstly, multiple solutions were all autoclaved and individually prepared. The medium was freshly prepared on the day of use. The appropriate antibiotic, chloramphenicol, was also added to the medium. After all preparations, 300 µL of each solution was aliquoted into Eppendorf tubes and stored at -20ºC.

  1. T-Base: (autoclaved)
    1. 2g of (NH4)2SO4
    2. 14g of K2HPO
    3. 6g of KH2PO4
    4. 1g of Na3citrate x 7H2O
    5. Fill up to 1 litre with dH2O
  2. SpC-Medium:
    1. 20mL of T-Base
    2. 0.2mL of 50% Glucose (w/v)
    3. 0.3mL of 1.2% MgSO4 (w/v)
    4. 0.4mL of 10% yeast extract (w/v)
    5. 0.5mL of 1% Casamino acids (w/v)

    For tryptophan-auxotrophic strains add 0.25 mL of tryptophan (8g/L, sterile filtered

  3. CaCl2 (100mM):
    1. 0.14702g of CaCl2 x H2O
    2. Fill up to 10mL with dH2O
  4. SpII-Medium:
    1. 200mL of T-Base
    2. 2mL of 50% Glucose (w/v)
    3. 14mL of 1.2% MgSO4 (w/v)
    4. 2mL of 10% yeast extract (w/v)
    5. 2mL of 1% Casamino acids (w/v)
    6. 1mL of CaCl2

    For tryptophan-auxotrophic strains add 2.5 mL of tryptophan (8g/L, sterile filtered).

  5. EGTA (100mM):
    1. 0.7607g of EGTA
    2. Fill up to 20 mL with dH2O

    For tryptophan-auxotrophic strains add 2.5 mL of tryptophan (8g/L, sterile filtered).

  6. SpII+EGTA:
    1. 10mL of T-Base
    2. 0.10mL of 50% Glucose (w/v)
    3. 0.70mL of 1.2% MgSO4
    4. 0.10mL of 10% yeast extract
    5. 0.10mL of 1% Casa Amino-Acids
    6. 0.22mL of EGTA

    For tryptophan-auxotrophic strains add 0.125 ml of tryptophan (8g/L, sterile filtered).

  1. In an Eppendorf tube (2 mL), 250 µL of competent B. subtilis cells were mixed with 250 µL of the SpII+EGTA solution.
  2. In another Eppendorf tube (2 mL), 20 µg of the plasmid was added and mixed with 300 µL of the suspension.
  3. The cell suspension was then incubated for 3 hours and at 37ºC and 350 rpm.
  4. Cell suspension than incubated for a second time at 21ºC and 350 rpm for 2 hours.
  5. Plates then prepared by spreading cells completely onto LB medium with chloramphenicol and incubated overnight at 37ºC.

Plasmid Purification (using GeneJET kit):

After plasmid growth and replication in the DH5α E. coli, the constructs were extracted through a series of minipreps. The method for these is detailed below.

Method/Protocol

All steps were performed at room temperature. All mentioned buffers/solutions are from the GeneJET miniprep kit.

  1. 5 mL of each of the overnight cultures were centrifuged for 10 minutes at 5000 rpm at 10ºC, and supernatant removed. The following steps were followed for all cultures.
  2. The pelleted cells were resuspended in 250 µL of the resuspension solution and the resuspension transferred to a microcentrifuge tube. This was done by pipetting up and down until no cell clumps remained.
  3. 250 µL of the Lysis Solution was added to the resuspension and the tube was inverted multiple times to ensure thorough mixing. This was noticed when solution became viscous and slightly clearer.
  4. 350 µL of the neutralization solution was then added and immediately gently mixed by tube inversion.
  5. The resulting solution was then centrifuged for 5 minutes at 10,000 rpm.
  6. The supernatant was then transferred to the supplied GeneJET spin column by pipetting as gently as possible so as to not disturb the precipitate.
  7. This was then centrifuged for 1 minute at 10,000 rpm. The flow through was discarded and column placed back onto collection tube.
  8. 500 µL of the Wash Solution was then added before centrifuging again for 1 minute at 10,000 rpm. The flow through was then discarded and column placed back onto collection tube. This was done twice.
  9. Column was centrifuged for one more minute at same rpm just to ensure all ethanol (from Wash Solution) had been removed.
  10. The spin column was then transferred onto a 1.5 mL microcentrifuge tube.
  11. 50 µL of the Elution Buffer was added to column centre to ensure plasmid elution. This was incubated at room temperature for one minute and then centrifuged for two minutes.
  12. The column was then discarded, and purified plasmid DNA was stored at -20ºC.
  13. DNA concentration was determined using the Qubit™ Fluorometric Quantitation kit.

DNA Concentration Determination (using Qubit™ fluorometric quantitation kit):

Method/Protocol

  1. Two Assay Tubes (thin wall, clear 0.5 mL PCR tubes) were set up for the standards and one for each sample to be measured.
  2. 200µL of the Qubit™ Working solution was prepared for each sample and standard. This was done by diluting the Qubit™ reagent 1:200 in Qubit™ Buffer.
  3. Assay tubes were all prepared using the table below from the Qubit™ reference card:
    Standards Assay Tube Sample Assay Tube
    Working Solution Volume to add 190 µL 180-199 µL
    Standard Solution Volume to add 10 µL -
    Sample Volume to add - 1-20 µL
    Total Assay tube Volume 200µL
  4. Vortex all tubes for 2-3 seconds.
  5. Incubate the tubes for 2 minutes at room temperature.
  6. Insert the tubes into the Qubit® 2.0 Fluorometer and take reading.

Protein Purification (using drip column):

Nickel Sepharose Media drip column was set up before experiment start on a clamp stand, column was stored in Ethanol. Both buffers were supplied and not prepared, compositions outlined in method. All proteins were Histone Tagged, allowing for Nickel column to be used as purification method.

Method/Protocol

For sample extraction;

  1. The bacteria were induced using IPTG.
  2. Approximately 4 hours after induction cells were frozen.
  3. Shortly before the purification experiment, the start cells were left to thaw and lysed by sonication at 5 micron amplitude for 10 cycles of 30 seconds on and 30 seconds off.
  4. The cells were then centrifuged and cell debris discarded, and supernatant labelled as column sample.
  5. The bung from the bottom of drip column was removed and EtOH was allowed to flow out of column and to drip onto the falcon tube and was then discarded. The bung was closed once more.
  6. 15 mL of Buffer A (50mM Tris HCl, pH 7.5, 0.5 M NaCl, 20 mM Imidazole) was carefully added to the top of the media and allowed to drip out until the buffer sat on top of media.
  7. Sample then carefully added to the top of the media, keeping any media disturbances to a minimum.
  8. The column bung was opened and sample allowed to slowly drip out and 3 mL fractions collected and labelled.
  9. To wash unbound sample, 15 mL of Buffer A was carefully added to the column top, which was then allowed to flow through and 5 mL fractions were then collected.
  10. To elute column bound protein, 15 mL of Buffer B (50mM Tris HCl, pH 7.5, 0.5 M NaCl, 500 mM Imidazole) was applied and allowed to drip, 1 mL fractions were collected in a deep well plate.
  11. To determine in which fraction the proteins were eluted, 100 µL of each fraction was into UV/VIS plate with Buffer A as a blank and blanking against the well was read with just Buffer A. A peak should be observed in fraction where protein was eluted.

SDS Page Gel/Electrophoresis

The denaturing gel electrophoresis was run using Nu-Bolt™ SDS-PAGE gel 10% Bis-Tris (ThermoFisher) manufacture’s instructions. The main aim for this experiment was to check if the size of the purified protein was the same as the protein aimed to overexpress, therefore using this as a verification if the engineered bacteria produced our desired product.

Method/Protocol

Firstly, 20X Bolt™ MOPS SDS Running Buffer (50 mM MOPS, 50 mM Tris Base, 0.1% SDS, 1 mM EDTA, pH 7.7) had to be prepared;

  1. The following reagents were dissolved in 400 mL ultrapure water:
    1. 104.6g of MOPS
    2. 60.6g of TrisBase
    3. 10.0g of SDS
    4. 3.0g of EDTA
  2. All reagents were mixed well, and volume was adjusted to 500 mL with ultrapure water. The sample preparation could then begin.
  3. To prepare the samples, 10 µL of the sample was transferred onto the Eppendorf tube and 10 µL of Bolt™ 4X LDS Sample Buffer and 4 µL of 10X Nu-Bolt™ PAGE Reducing Agent. Make up volume to 40 µL using deionized water.
  4. Samples were heated at 70ºC for 10 minutes.
  5. As the samples were heated, the buffers were prepared by adding 20 mL of 20X Bolt™ MOPS SDS Running Buffer to 380 mL of deionized water, making 1X SDS Running Buffer. Due to sample reduction, 1 mL of Bolt™ Antioxidant to 400 mL 1X SDS Running Buffer.
  6. To prepare the gel, the gel wells were washed thrice (with Running Buffer) then the gel chambers were filled with 1X SDS Running Buffe. The gel was then placed in gel tank.
  7. The ladder was then loaded by 10 µL of the ladder into well 1, and the remaining wells were filled with 40 µL of the sample. The gel was then left to run for 32 minutes at 200 V.
  8. To stain the gel, Generon Quick Coomassie Stain was used and 25 µL of it was incubated with the gel for 1 hour.
  9. To destain gel, it was incubated with 100 mL of distilled water for 1 hour.

Western Blot

The Western Blot was performed for SDS page visualization and towards the same aim, as a verification if the engineered bacteria produced overexpressed protein. The primary antibody used was an anti His-tag antibody raised in Mouse, and the secondary antibody is an anti-Mouse antibody bound to alkaline phosphate (AP) raised in Goat. The detection method used BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/ nitro blue tetrazolium) tablets, which were acquired from SigmaFast. An insoluble substrate was used for the detection of alkaline phosphatase.

Method/Protocol

  1. SDS PAGE Gel was run using the above protocol.
  2. The preparation of 30 mL 1X iBind™ Solution was required for each run. This was done by mixing the following:
    1. 6.0mL of iBind™ 5X Buffer
    2. 0.3mL of iBind™ 100X Additive
    3. 23.7mL of distilled water
  3. Next, the PVDF membrane was prepared by trimming it to 9 cm x 9 cm, and then pre-activated in 100% methanol and rinsed in distilled water. The blotted membrane was then submerged (protein-side up) in 5 mL of 1X iBind™ Solution.
  4. 2 mL of Primary antibiotic solution was then prepared for each run by mixing 2 mL of 1X iBind™ Solution with 2 µL anti his-tag raised in mouse antibody.
  5. 2 mL of Secondary antibiotic solution was then prepared for each run by mixing 2 mL of 1X iBind™ Solution with 2 µL anti his-mouse raised in goat-alkaline phosphate conjugated antibody.
  6. iBind™ run could then start being prepared by adding 6 mL of 1x iBind™ Solution evenly across the flow region of plastic gel box.
  7. iBind™ card was then placed into the box and 5 mL of iBind™ Solution pipetted evenly across flow region.
  8. 1 mL of iBind™ Solution was then pipetted to the centre of the card.
  9. The prepared membrane then placed upside down (protein-side down and low molecular weight region closest to the stack) on top of pooled solution. Membrane was not allowed to touch the stack and no part of it was directly under any of the wells.
  10. The blotting roller was then used to remove any air bubbles formed.
  11. iBind™ machine was then closed, and to the wells on top the following was added:
    Well 1 2 mL diluted primary antibody
    Well 2 2 mL 1X iBind™ Solution
    Well 3 2 mL diluted secondary antibody
    Well 4 6 mL 1X iBind™ Solution
  12. Window cover was placed, and apparatus closed and left for 3 hours.
  13. After the end of incubation period, the well cover was slightly raised to verify Well 4 was completely empty- indicating run is over. If this was not observed, iBind™ was left to incubate in 10-minute increments until the run was over.
  14. Membrane was then removed from the machine and rinsed twice in 20 mL of distilled water for 2 minutes.
  15. Detection was then run by dissolving one tablet of SigmaFast BCIP/NBT in 10 mL of ddH2O, 2.5 mL of resulting solution was added evenly across membrane. This was incubated for 5 minutes.
  16. Excess solution blotted with filter paper, but the membrane was not allowed to dry out.
  17. Visualization was performed in lid until ladder developed.
  18. Membrane was then stored in ddH2O.

Agarose/Hydrogel Experiment:

These experiments were carried out to investigate the carbon dioxide diffusion onto a hydrogel mesh. This data was to be later used and implemented on our project printer application. A colour change can be observed due to the formation of carbonic acid, a product of the reaction of the CO2 with the hydrogel.

Method/Protocol

  1. Different quantities of agarose were mixed onto 300 mL of milli-Q water in a conical flask to compose the three different concentrations, outlined below. The next steps were then followed for every repeat.
    Agarose Concentration Agarose Quantity Added CO2 Exposure
    0.1% 3g 24hrs
    1% 30g 24hrs
    1% 30g Not exposed
  2. This was then swirled and put into a microwave for 20 second intervals with swirling in between, to make sure agarose has dissolved.
  3. Once the agarose was fully dissolved, the flask was left to cool down.
  4. As the agarose cooled, 3 glass flasks were washed with hot water and covered with parafilm.
  5. Once cooled, 100 mL of agarose solution was poured onto each flask. Then, 1000 µL of universal indicator solution was pipetted onto the first flask – that was quickly covered with parafilm.
  6. Step 5 was repeated for two remaining flasks.
  7. Once all three flasks were prepared, they were placed in the fridge for 1 hour.
  8. Flasks were removed from the fridge – one was chosen as control and parafilm left on, the other two had their parafilm removed.
  9. All flasks were then exposed to CO2 for 24 hours in an incubator.
  10. The time-lapse on the Raspberry Pi™ camera was then started.
  11. After the 24hrs passed, all flasks were removed from the incubator and colour change observed.

Exeter iGEM 2020

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