Team:BOKU-Vienna/Experiments

Experiments



    Experiments

    GG assembly

    Golden Gate Cloning is one of the easiest cloning methods as it provides a fast one-step reaction which leaves no cloning sites in the final construct, cutting out unnecessary base pairs, therefore making it irreversible. The restriction enzymes cut off the fusion sites of inserts and backbones, leaving matching overhangs to fuse. Typically, promotors, coding sequences and terminators are purchased as gBlocks with the appropriate fusion sites and are then individually cloned into the first backbone, backbone 1. Afterwards, promotors, coding sequences and terminators are fused into backbone 2. In the last step, multiple promotor-coding sequence-terminator blocks can be cloned into a single final backbone.

    Fusion itself is done using a Thermocycler, often overnight. The assembly mix contains the inserts, backbones, ATP, DNA ligase, buffer, and water. Following the assembly, DNA is transformed into competent cells (e.g., using heat shocking or electroporation). Backbones contain an antibiotic resistance and cells are plated on media with respective antibiotics for selection. After incubation the grown colonies can be picked, and DNA is extracted using a Miniprep system. Identity of the plasmid and inserts can be confirmed via sequencing.Thereafter, the next cloning step may be performed by using the extracted and sequenced DNA as insert.

    In silico planning of inserts must be done carefully. Appropriate fusion sites are needed, and sequences need to be optimized to not contain unwanted cutting sites (e.g., having a restriction site used in one of the ensembles inside of the coding sequence).However, one is rewarded with an easy, efficient, and reliable cloning system.

    Protocols

    GG assembly

    The gBlocks were designed by our research team and ordered from IDT. Enzymes, buffers as well as the T4-Ligase were purchased from New England Biolabs. The backbones and ATP were kindly obtained from our supervisors.

    Vecors & Inserts

    Backbone 1_Fusion site 1-2 Promotors
    Backbone 1_Fusion site 2-3 Enzymes
    Backbone 1_Fusion site 3-4 Terminator
    Backbone 2_Fusion site A-B Promotor + Enzyme 1 + Terminator
    Backbone 2_Fusion site B-C Promotor + Enzyme 2 + Terminator
    Backbone 2_Fusion site C-D Promotor + Enzyme 3 + Terminator
    Backbone 3_Fusion site A-B P + E1 + T
    Backbone 3_Fusion site A-C P + E1 + T & P + E2 + T
    Backbone 3_Fusion site A-D P + E1 + T & P + E2 + T & P + E3 + T

    Rescrition enzymes

    Backbone 1 for BsaI
    Backbone 2 for BbsI
    Backbone 3 for BsaI

    Reaction mix

    Total reaction volume 20 µL
    Vector 1 µL
    Fragment 1 µL each
    Restriction enzyme 1 µL
    CutSmart buffer 10x 2 µL
    ATP 10x 2 µL
    T4-Ligase (1:10 diluted 40 U) 2.5 µL
    RO-H2O ad 20 µL

    All is added into a PCR tube. Enzymes are added last and kept in cooling racks until placed in Thermocycler.

    Golden Gate assembly program for Thermocycler: LONG

    Cutting 37 °C 5 min 45 cycles
    Ligation of sticky ends 16 °C 2:30 min
    Fragment linearization 50 °C 5 min
    Enzyme deactivation 80 °C 10 min
    Cool down 25 °C 15 min

    Golden Gate assembly program for Thermocycler: SHORT

    Cutting 37 °C 5 min 40 cycles
    Ligation of sticky ends 16 °C 2:30 min
    Fragment linerarization 50 °C 5 min
    Enzyme deactivation 80 °C 10 min
    Cool down 25 °C 15 min

    Afterwards a post-digest is performed to degrade any backbones without inserts. For this 0.5 µL of the appropriate restriction enzyme is added to each reaction tube and placed in the Thermocycler.

    Post digest program for Thermocycler

    Cutting 37 °C 30 min
    Enzyme deactivation 65 °C 20 min
    Cool down 25 °C 1 s

    Transformation

    Preparation of chemically competent E.coli

    E.coli strain DH10B is inoculated from a glycerol stock onto an LB Agar plate. The inoculum is streaked on the plate using an inoculation loop to obtain individual colonies. The plate is incubated overnight at 37 °C.
    A flask with 10 mL of LB medium is inoculated with a single colony and incubated overnight in a shaker-incubator (37 °C, shaking 180 rpm). The following day, 2 mL of this culture is transferred to a 2 L shake flask containing 200 mL LB medium and incubated for about 2.5 hours until OD600 reaches 0.6. It is checked continuously, first after 1.5 hours. Blank water (or LB medium) is used before every measurement and the cell mixture is not diluted. The cells are cooled down on ice for 10 min and then pelleted in a centrifuge for 5 min at 4500 rpm (4000 × g) at 4 °C. The cells are resuspended in 80 mL volume of ice-cold TFB1. The centrifugation step is repeated. The pellet is resuspended in 8 mL of ice-cold TFB2. The cells are aliquoted 100 μL per tube and stored at −80 °C until used.

    Transformation in E.coli

    1. Frozen chemically component cells (100 µL per tube) are thawed on ice.
    2. 10 µL of GG product is added to the cells, cells and DNA are then gently mixed.
    3. Let the cells-DNA-Mix stand on ice for 30 min.
    4. Cells and DNA-mix are heat shocked for 90 s at 42 °C (exact time noted).
    5. The cells are allowed to recover on ice for 5 min.
    6. 1 mL of LB Medium without antibiotics is added and the cells are incubated at 37 °C for:
      • 90 min (if Kanamycin is used for backbone 1)
      • 30 min(if Ampicillin is used for backbone 2)
      • 60 min(if Erythromycin is used for backbone 3)
    7. Cells are being distributed with a Drigalski spatula on the Agar plates at different concentrations:
      • 1 µL (add 90 µL LB Medium to the plate first)
      • 10 µL (add 90 µL LB Medium to the plate first)
      • 100 µL
      • Rest (centrifuge, discard all but 100µL of supernatant, resuspend and add 100 µL to plate)
    8. The plates incubated at 37 °C overnight.

    Preparation of electrocompetent L.plantarum

    1. Electrocompetent L.plantarum strain NC8 is always freshly prepared before a transformation.
    2. A pre-culture is prepared the day before in 20 mL MRS-medium with a pH of 6.2 + 2 % glucose and placed in an anaerobic jar at 30 °C, 180rpm overnight.
    3. The next day OD600 is measured, and it is calculated how much of the pre-culture needs to be added to another flask with 500 mL of MRS-medium + 2 % glucose + 1 % glycine to equal an OD600 of 0.3.
    4. The main culture is then grown at 30 °C, 0 rpm until OD600 of 0.6 is measured.
    5. The culture is then centrifuged 10 min at 4000 rpm and 4 °C. All buffers are kept on ice throughout.
    6. The pellet is suspended in 100 mL washing buffer and washed 5 times, each centrifugation step is 10min at 4000rpm and 4 °C.
    7. The pellet is then resuspended in 1 mL of electroporation buffer and the OD600 is measured.

    Transformation in L.plantarum:

    1. 80 µL of the electrocompetent cells are mixed with 20 µL DNA.
    2. Electroporation is done at 2500 V, 200 Ω, 25 µF in 4 mm cuvettes à 6250 V/cm
    3. Afterwards the cells are diluted with 900 µL MRS-medium + 2 % glucose + 0.3 M sucrose and incubated at 30 °C for 3h and 180 rpm.
    4. The cells are plated on MRS Agar + 2 % glucose + 2.5 µg/mL erythromycin plates with concentrations of 50, 100, 200, 300 µL and incubated at 30 °C for 3 to 5 days.

    Colony PCR

    Reaction mix

    Total reaction volume 10 µL
    AccuStart II PCR SuperMix 5 µL (hot-start polymerase)
    Forward sequencing primer 1 µL
    Backward sequencing primer 1 µL
    RO-H2O + picked colony 3 µL

    All is added into a PCR tube and placed in the Thermocycler.

    PCR amplification program for Cycler

    Disintegration & Activation 94 °C 5 min
    94 °C 20 s35 cycles
    63 °C 25 s
    68 °C 2:10 min
    Cool down 25 °C 2 s

    Promising clones are selected via agarose gel electrophoresis (1 % agarose gel, 130 V, 1 h) and an overnight culture is prepared for minipreps.

    PCR amplification (instead of transformation of BB3 in E.coli)

    Reaction mix

    Total reaction volume 50 µL
    DNA template 5 µL
    AccuStart II PCR SuperMix 25 µL
    Forward sequencing primer 5 µL
    Backward sequencing primer 5 µL
    RO-H2O 10 µL

    All is added into a PCR tube and placed in the Thermocycler.

    PCR amplification program for Cycler

    Disintegration & Activation 94 °C 5 min
    94 °C 20 s 35 cycles
    63 °C 25 s
    68 °C 2:10 min
    Cool down 25 °C 2 s

    Promising clones are selected via a preparative agarose gel electrophoresis (1 % or 1.5 % agarose gel, 130 V, 1 h), the appropriate bands are cut out and purified. The concentration is measured via Nanodrop, and the clones are sequenced for confirmation.

    PCR for construction of BB3

    Reaction mix

    Total reaction volume 50 µL
    DNA template 5 µL
    Q5® High-Fidelity 2X Master Mix 25 µL
    Forward sequencing primer 2.5 µL
    Backward sequencing primer 2.5 µL
    Nuclease free RO-H2O 15 µL

    All is added into a PCR tube and placed in the Thermocycler.

    Elongation Temp + 25 s Annealing Temp:

    Construct 1, 2 & 3 50 sec + 25 sec = 75 sec
    Construct4 & 5 24 sec + 25 sec = 49 sec

    PCR amplification program for Cycler for construct c1-3 and c4-5:

    Denaturation 98 °C 30 s
    98 °C 10 s 35 cycles
    72 °C 75s/49s
    72 °C 10 min
    Cool down 10 °C 2 min

    Promising clones are selected via a preparative agarose gel electrophoresis (1 % or 1.5 % agarose gel, 130 V, 1 h), the appropriate bands are cut out and purified. The concentration is measured via Nanodrop, and the clones are sequenced for confirmation.

    Miniprep & Gel Purification

    According to protocol of the following Kits:

    Miniprep for E. coli

    Monarch® Plasmid Miniprep Kit by New England Biolabs
    Hi Yield® Plasmid Mini Kit by SLG

    Deviations:

    • 2 mL culture used
    • Extra step for sequencing done
    • Eluted twice with 50 µL of 50 °C warm 10 mM TRIS

    Deviations from MIDI prep of L. diolivrans for Mini prep of L.plantarum:

    • 5 mL culture, harvest: 3200 rpm, 30 min
    • Add 0.4 mL 5M LiCl (6.36 g/30 mL) - (2 mL 10M LiCl stock)
    • Incubation: 37 °C, 180 rpm, 20 min
    • Transfer mixture into 2 mL Eppis and centrifuge at 13000 rpm for 2 min
    • Dissolve pellet in 1 mL AD
    • Add 1 mL AD
    • Centrifuge at 3200 rpm, 10 min
    • Add Prep Mix (1 g Lysozyme + 500 µL RNAseA + 200 µL Mutanolysin-stock dissolved in 50 mL P1) and incubate at 37 °C, 180 rpm, 50 min
    • Continue according to the blue protocol unit step 4
    • Add 5 mL P1
    • Add 10 mL P2
    • Add 10 mL P3
    • Continue according to the grey protocol from step 6 on
    • Centrifugation
    • Add TRIS and incubate at RT o/n

    Gel purification

    ReliaPrep™ DNA Clean-Up and Concentration System by Promega
    Wizard® SV Gel and PCR Clean-Up System by Promega

    Deviations:

    • Eluted twice with 15 µL of 50 °C warm 10 mM TRIS

    Restriction digest

    Reaction mix

    Total reaction volume 10 µL
    DNA (Miniprep) 600 ng diluted with water to equal 20 µL
    Enzyme 1 0.5 µL
    Enzyme 2 (if necessary) 0.5 µL
    Buffer 2 µL

    Samples are put on 37 °C for 1 h. Promising clones are selected via agarose gel electrophoresis (1 % agarose gel, 130 V, 1 h) and subsequently confirmed via sequencing.

    Media

    LB medium & LB Agar for E.coli

    Peptone Hy-Soy 40.00 g +/- 1.00 g
    Yeast extract 20.00 g +/- 0.50 g
    NaCl 20.00 g +/- 0.50 g
    RO-H2O ad 4000.00 g
    • Adjust pH to 7.4 - 7.6 with 4N or 8N NaOH
    • Bottle 500 g in 500 mL Schott bottles
    • Sterilize by autoclaving for 20 minutes at 121°C
    • For LB Agar add 10.00 g +/- 0.25 g of Agar-Agar per 500mL Schott bottle

    MRS medium & MRS Agar for L. plantarum:

    RO-H2O 2000.00 g +/- 50.00 g
    Casein peptone 40.00 g +/- 1.00 g
    Meat extract 40.00 g +/- 1.00 g
    Yeast extract 20.00 g +/- 0.50 g
    Tween 80 4.00 g +/- 0.10 g
    K2HPO4 8.00 g +/- 0.20 g
    Sodium acetate 20.00 g +/- 0.50 g
    Tri-sodium citrate dihydrate 10.40 g +/- 0.26 g
    (NH4)2HPO4 4.68 g +/- 0.12 g
    MgSO4 + 7 H2O 0.80 g +/- 0.02 g
    MnSO4 +H2O 0.20 g +/- 0.01 g
    RO-H2O ad 3600.00 g
    • Adjust pH to 5.7 – 6.2 with HCl
    • Bottle 500 g in 500 mL Schott bottles
    • Sterilize by autoclaving for 20 minutes at 121°C
    • Before use: Add 50 mL of 10 x carbon source (e.g., 10x glucose, 10x glycerol) per bottle
    • for MRS Agar add 10.00 g +/- 0.25 g of agar-agar per 500mL Schott bottle

    Buffers for L. plantarum electroporation:

    • Washing buffer
      0.3 M sucrose

    • Electroporation buffer
      272 mM sucrose
      7 mM sodium phosphate

    • Resuspension buffer
      MRS with a pH of 6.2 + 2 % glucose
      0.3 M sucrose

    Experiments

    Since preclinical and clinical trials were out of scope for the iGEM competition, we were looking for a different way to test the biocompatibility of our scaffold. We got in touch with Professor Peter Ertl from the Cell Chip Group at the Technical University of Vienna, who suggested that his organ-on-a-chip technologies might be a good alternative, as they can mimic membrane barriers and fluidic systems which play an important role in the way how drugs interact with the body. He gave us the opportunity to use his lab and receive guidance from Doris Roth for our experiments. She, however, advised us against using a gut-on-a-chip for several reasons: One of the most important being that it can take several months until one gets a working and inoculated chip. Additionally, organs-on-a-chip are too small to test a macroscopic capsule. In the end, we settled on going the classic route of an in vitro cytotoxicity test with Caco-2 cells and, optionally, a transwell test to see if the polymer can pass through membranes.

    Cytotoxicity tests in cell culture are well established and commonly used for the assessment of the biocompatibility of all kinds of materials. Since our capsule would only get in contact with intestinal membranes, the Cell Chip Group recommended Caco-2, an immortalized human colorectal adenocarcinoma cell line. This cell line is widely used as an in vitro model for the mucosa of the small intestine and used for the Cell Chip Group’s gut-on-a-chip.

    To test our scaffold, we decided to do the resazurin or MTT assay, which is one of the most popular and widely used cell viability tests. It is an excellent way to assess the toxicity of a material in vitro. The assay is based on the reduction of oxidized non-fluorescent blue resazurin to a red fluorescent dye (resorufin) in cellular mitochondria. Therefore, a fluorescent signal indicates live cells. The proper seeding density must be adjusted to the respective cell line. Changes of incubation times with the test material as well as with the resazurin solution strongly affect the results, so they should be chosen wisely. Four hours of incubation with the resazurin solution are often regarded as ideal.

    Cells used for assays should be active and healthy cultures (generally, they should be split at least twice to make sure they have recovered from being frozen) before using them in an assay. They should also not have been passaged too often, since at some point, genomic abnormalities can occur, or they start to grow more slowly.

    Since our test material, chitosan, is not easily soluble in water at neutral pH but highly soluble in an acidic solution, we decided to decrease the pH to 6.3, which should be tolerated by Caco-2 cells. Here, we planned to test both the well-characterized chitosan, as well as our thiolated chitosan. Additionally, we thought about applying a fine suspension of powdered, lyophilized chitosan and thiolated chitosan, in case we would not manage to dissolve the polymers. Adding the whole capsule is no option since this might kill the cells or the material would get overgrown, making the results unusable. We chose to test a concentration of 0.25 % (w/v) since higher concentrations would likely be too viscous.

    Protocols

    Caco-2 - Thawing

    Introduction

    Thawing of frozen Caco-2 stocks for culture

    Materials

    • T25 flask
    • 10 mL Caco-2 medium (total)
      • 80 % DMEM
      • 20 % FCS
      • 1 % streptomycin/penicillin (added on top)

    Procedure

    Thawing:

    1. Preheat medium to 37 °C.
    2. Quickly thaw cells in your hand.
    3. Add 4-5 mL preheated medium to the flask and add the cells from the cryovial. Gently mix by pipetting up and down.
    4. Place the flask in the incubator. Criss-cross to distribute the cells evenly.

    Change of Medium:

    1. Place the flask in the incubator. Criss-cross to distribute the cells evenly.

    Caco-2 - Maintenance

    Introduction

    Medium needs to be changed on Monday, Wednesday, and Friday. At 80-90 % confluency, cells need to be split (see protocol "Caco-2 - Splitting").

    Materials

    Caco-2 medium

    • 80 % DMEM
    • 20 % FCS
    • 1 % streptomycin/penicillin (added on top)

    Procedure

    Medium exchange:

    1. Preheat fresh medium to 37 °C.
    2. Remove old medium from the side opposite to the cells as to not accidentally detach cells.
    3. Add fresh medium, again at the opposite site.
      • For T25 flasks: 4-5 mL
      • For T75 flasks: 15 mL
    4. Place flask back in the incubator.

    Caco-2 - Splitting (Passage)

    Introduction

    If the cells are at 80-90 % confluency, they need to be split. The splitting ratio depends on how fast we want our cells to be ready for splitting again. The usual ratio is 1:3 for Caco-2 cells. This means that 1/3 of the cells are transferred into the new flasks. Several flasks can be inoculated as needed.

    Materials

    For T25 flasks:

    • ~10 mL Caco-2 medium
    • 2 mL trypsin
    • 10-15 mL PBS (without calcium)

    For T75 flasks:

    • ~25 mL Caco-2 medium
    • 3 mL trypsin
    • 20-30 mL PBS (without calcium)

    Procedure

    T25 flasks:

    1. Preheat medium, PBS and trypsin to 37 °C.
    2. Remove old medium.
    3. Gently wash 2-3 x with 4 mL PBS to remove all remaining medium.
    4. Add 2 mL trypsin.
    5. Incubate for max. 5 min at 37°C until cells are detached.
    6. Stop the reaction with 3 mL medium, washing down cells from the flask surface and transfer them into a Falcon tube.
    7. Count cells if needed.
    8. Centrifuge cells for 5 min at 1500 rpm.
    9. Remove supernatant and resuspend cells in fresh medium.
    10. Split cells 1:3 or as needed into new flasks to a total volume of 4-5 mL per flask. Don't forget to write down the new passage number.

    T75 flasks:

    1. Preheat medium, PBS and trypsin to 37 °C.
    2. Remove old medium.
    3. Gently wash 2-3 x with 10 mL PBS to remove all remaining medium.
    4. Add 3 mL trypsin.
    5. Incubate for max. 5 min at 37 °C until cells are detached.
    6. Stop the reaction with 7 mL medium, washing down cells from the flask surface and transfer them into a Falcon tube.
    7. Count cells if needed
    8. Centrifuge cells for 5 min at 1500 rpm.
    9. Remove supernatant and resuspend cells in fresh medium.
    10. Split cells 1:3 or as needed into new flasks to a total volume of 15 mL per flask. Don't forget to write down the new passage number.

    Caco-2 - Seeding

    Introduction

    Seeding of Caco-2 cells into 96-well plates for live/dead stainings to test biocompatibility or transwells for barrier experiments. Cells should have been split twice before they are used for an experiment and be grown in a T75 flask to ensure that there are enough cells for the experiment which are healthy and growing well.

    Materials

    • Caco-2 medium
      • 80 % DMEM
      • 20 % FCS
      • 1 % streptomycin/penicillin (added on top)
    • Trypsin
      • PBS (without calcium)
      • 96-well plates OR 24-well plates and transwells

    Procedure

    Cell Detachment:

    1. Cells should have been split at least twice before they are used for an experiment.
    2. Preheat medium, PBS and trypsin to 37 °C.
    3. Remove old medium.
    4. Gently wash 2-3 x with 10 mL PBS to remove all medium.
    5. Add 3 mL trypsin.
    6. Incubate for max. 5 min until cells are detached.
    7. Stop the reaction with 7 mL medium, washing down the cells from the flask surface.

    Adjusting cell density:

    1. Count cells and calculate dilution. Each well should contain 8000 cells; each well holds 200 µL volume.
    2. Centrifuge cells for 5 min at 1500 rpm.
    3. Remove supernatant and resuspend cell pellet in fresh, preheated medium to adjust cell density to 8000 cells/200 µL.

    Seeding - 96 well plates:

    1. Seed 200 µL cell suspension per well, leaving the outer rows empty or filling them with PBS to reduce evaporation.
    2. Incubate at 37 °C.
    3. Change medium after 4 h.
    4. Grow overnight before starting the experiment.

    Seeding - Transwell plates:

    1. Add transwells to each well, leaving the top and bottom row empty or filling them with PBS to reduce evaporation.
    2. Outer well: 400-500 µL
    3. Inner well: 200 µL
    4. Adjust outer surface level to avoid hydrostatic pressure.
    5. Incubate at 37 °C.
    6. Change medium after 4 h.

    Resazurin assay for cytotoxicity testing on Caco-2 cells

    Unfortunately, due to limited time in the wet lab this experiment was not done. However, we decided to include this protocol in our Wiki to potentially help future iGEM-Teams:

    The resazurin or MTT assay is one of the most popular and widely used cell viability tests. It is an excellent way to assess the toxicity of a material in vitro. The assay is based on the reduction of oxidized non-fluorescent blue resazurin to a red fluorescent dye (resorufin) in cellular mitochondria. Therefore, a fluorescent signal indicates live cells. The proper seeding density must be adjusted to the respective cell line. Changes of incubation times with the test material as well as with the resazurin solution strongly affect the results, so they should be chosen wisely. Four hours of incubation with the resazurin solution are often regarded as ideal.

    Cells used for assays should be in active culture and healthy (generally, they should be split at least twice to make sure they have recovered from being frozen) before using them in an assay. They should also not have been passaged too often, since at some point, genomic abnormalities can occur, or they start to grow more slowly.

    Since our test material, chitosan, is not easily soluble in water at neutral pH but highly soluble in an acidic solution, we decided to decrease the pH to 6.3, which should be tolerated by Caco-2 cells. Here, we planned to test both the well-characterized chitosan, as well as our thiolated chitosan. Additionally, we thought about applying a fine suspension of powdered, lyophilized chitosan and thiolated chitosan, in case we would not manage to dissolve the polymers. Adding the whole capsule is no option, since this might kill the cells or the material would get overgrown, making the results unusable. We chose to test a concentration of 0.25 % (w/v) since higher concentrations would likely be too viscous.

    • are seeded in a 96-well plate at a density of 2 x 104 cells per well in a final volume of 200 µL Caco-2 culture medium (see Seeding protocol).
    • The cells containing the seeded cells are stored in the incubator (37 °C, 5 % CO2) until they have created a monolayer. This should take 24 h to max. 2 days.
    • The samples are prepared at a final concentration of 0.25 % (w/v) in Caco-2 culture medium.
    • Cells are washed 2-3 times with 100 µL PBS before test solutions are applied at a volume of 100 µL. The same volume of Caco-2 medium is applied to serve as a positive control, and Triton X 100 at a concentration of 4 % is added as a negative control. All samples are done at least in triplicates.
    • The cells are again incubated for 24 h.
    • Following the 24h of incubation, test and control solutions are removed, and after washing the cells twice with PBS, 100 µL of a 5 % (w/v) resazurin solution is applied.
    • Cells are incubated for 4 h.
    • The fluorescence intensity of each well is determined at an excitation wavelength of 540 nm and an emission wavelength of 590 nm using a microplate reader.
    • Cell viability is calculated according to the following equation:

      Cell viability [%] = (intensity of sample – intensity of negative control)/(intensity of positive control – intensity of negative control) * 100

    Experiments

    Our scaffold is one of the main parts of our project Friendzyme.

    Its main roles are:

    1. Acting as a biocontainer
    2. Providing mucadhesion
    3. Protection of the cells against harsh conditions

    The biocontainer

    The biocontainer is an essential part of our biosafety concept which ensures that the GMOs we want to use do not proliferate freely and thus pose a threat to the patient or the environment.

    Our scaffold is designed in a way to keep the modified Lactobacillus separate from other bacteria in the gut. The pore size is chosen in a range that our Lactobacillus cannot escape through the mesh, but small molecules and secreted proteins can freely diffuse in and out. The modified lactobacilli are living off the nutrients provided by the intestinal lumen and in return secrete fructan-degrading enzymes that exit the scaffold and decompose the fructo-oligosaccharides before they can cause symptoms in the lower intestine (see Protocols: Enzyme Escape, and Cell Escape)

    Mucoadhesion

    The surface of our scaffold is designed to be mucoadhesive by the addition of thiolated chitosan to the polymer solution that forms the scaffold.

    It will stick to the mucosa of the gut after passing through the stomach by forming disulfide bonds with the mucosa, which contains a lot of cysteines and thus is abundantly interlinked by this type of strong, covalent bond.

    Its mucoadhesive properties will allow the scaffold to stay attached to the surface of the intestine and release the secrete there for a prolonged timespan. By that, we hope to create a novel system that - compared to classical enzyme therapy - will be based on living cells that continuously provide a constant supply of fructan-degrading enzymes and thus eliminate the need for frequent intake of medication (see Protocol: Thiolation).

    Protection

    It has also been shown that a scaffold made from cellulose sulfate and poly-DADMAC can protect cells from the aggressive environment of the gastric fluid. Although it is planned that administration of our scaffold particles will happen in the form of a coated pill this feature could potentially be beneficial.

    Our approach

    Our scaffold is composed of two biocompatible polymer solutions that upon mixing instantly form a polyelectrolyte complex due to their charge. The concept is based on the product “Cell in a Box” (Austrianova) and was modified in order to additionally achieve mucoadhesion.

    The cells are suspended in a solution containing negatively charged cellulose sulfate, one of the three main components of our biocontainer. Due to time restraints, we did not finish fine-tuning the synthesized cellulose sulfate and used for further tests the cellulose sulfate from the Cell-in-a-Box kit (see Protocol: Synthesis of Cellulose Sulfate).

    On the surface of the droplet where the two solutions come in contact the polyelectrolyte-complex forms spontaneously, yielding a stable gel-like shell with a certain mesh size, and the bacteria on the inside of the droplet are encapsulated within it.

    This solution is then dripped into a bath that contains positively charged Polydiallyldimethylammonium chloride (polyDADMAC), as well as thiolated chitosan to provide the mucoadhesive properties (see Protocol: own solution).

    Safety considerations

    The overall size of our scaffold capsules is considerably larger than the mucus mesh network spacing (100-200 nm). Thus, it will not be able to penetrate the mucus layer.

    References

    Gunzburg, W. H., Aung, M. M., Toa, P., Ng, S., Read, E., Tan, W. J., Brandtner, E. M., Dangerfield, J., & Salmons, B. (2020). Efficient protection of microorganisms for delivery to the intestinal tract by cellulose sulphate encapsulation. Microbial cell factories, 19(1), 216. https://doi.org/10.1186/s12934-020-01465-3

    Hussain Asim, M., Nazir, I., Jalil, A., Matuszczak, B., & Bernkop-Schnürch, A. (2020). Tetradeca-thiolated cyclodextrins: Highly mucoadhesive and in-situ gelling oligomers with prolonged mucosal adhesion. International journal of pharmaceutics, 577, 119040. https://doi.org/10.1016/j.ijpharm.2020.119040

    Protocols

    Enzyme escape

    Materials

    • Bovine Serum Albumin (BSA), Sigma-Aldrich
    • Nanodrop
    • PBS
    • Austrianova Cell-in-a-Box® Kit
    • 12 eprouvettes
    • 3 ampoules

    Method

    1. Prepare 12 eprouvettes with 3 mL kit 2 solution (dilute like described in package insert)
    2. Prepare 3 ampoules with 150 µL PBS each
    3. Add 2.5 mg of BSA to 120 µL kit 1 solution
    4. Vortex
    5. One drop in each eprouvette
    6. Remove capsules after 10 min and put 5 capsules in each ampoule
      • Place ampoules on shaker (low speed) at room temperature
    7. Take samples of 10 µL supernatant of each ampoule at every timepoint
      • t = 0 h; 0.5 h; 1 h; 3 h; 24 h
    8. Measure samples with Nanodrop (measurement for protein A280)
      • Measure every sample 3x (3x2 µL)
      • Concentration should increase
    9. Calibration line concentrations:
      • 0.0625 mg/mL; 0.125 mg/mL; 0.25 mg/mL; 0.5mg/mL; 1.0mg/mL; 1.4mg/mL
      • Blank: supernatant of empty capsule in 150 µL PBS

    Cell escape assay

    Purpose

    The purpose of this assay is to demonstrate the functionality of the polymeric scaffold. It can be used to assess whether the pore size is adequate to retain the cells on the inside of the capsule and prevent them from escaping into the surrounding medium.

    Material

    • Austrianova Cell-in-a-Box® kit
    • 1x Phosphate Buffered Saline (PBS)
    • Glass eprouvettes
    • LB-Agar plates (with antibiotic added if necessary)
    • Small glass tubes (we used HPLC tubes)
    • 1 mL syringes (Braun Sterican®)
    • Blunt-ended syringe needle, diameter 0.4 mm (Braun)
    • Small metal spatula

    1. Experiment set-up

      • Prepare 15 eprouvettes each containing 3 mL kit solution 2 (dilute according to manual)
      • Prepare three glass tubes containing 100 µL PBS/capsule (final volume depends on how many capsules should be put into the tube)

    2. Preparation of the cell solution:

      1. To Spike 150 µL of kit 1 with 2*108 E.coli cells/mL
        • Measure the OD of the cell culture
        • Calculate the right dilution for E.coli and centrifuge the cells for 7 minutes
        • Remove the supernatant and add the 150 µL of solution 1 to the cell pellet
        • Vortex, then let settle until most of the air bubbles have vanished

    3. Encapsulation

      1. Take up the cell solution with the syringe needle. NOTE: The solution is quite viscous, so care should be taken when aspirating in order to avoid any air bubbles
      2. One drop into each eprouvette from a height of about 10 cm – capsule formation should occur instantly and the capsule sink to the ground. NOTE: Avoid air bubbles!
      3. After 10 min of hardening time, remove the capsules from the hardening bath with a spatula and transfer them into a prepared glass vial (see step 1B) and put the vial on a 360° shaker (approximately 50 rpm)
      4. Draw samples of 100 µL supernatant and one capsule of each ampoule at every timepoint
        • 0 h; 0.5 h; 1 h; 2 h and 24 h
        • Plate (capsules were ruptured before plating using a sterile toothpick)
        • Incubate at 37 °C for 24 h

      Expected results: Only plates of capsules should be overgrown. This shows that cells survive within the capsule but did not escape into the supernatant.

    Thiolation

    Altered microwave method (1)(2)(3)

    Reagents

    • 1 g chitosan, low molecular weight (Sigma Aldrich)
    • 1.4 g thiourea (Sigma Aldrich)
    • 50 mL 1 % acetic acid
    • 1.6 mL 37 % HCl
    • 1 M NaOH
    • Aceton

    Method

    • Solve 1 g chitosan (low viscosity) in 50 mL 1 % acetic acid in a 100 mL round bottom flask with a magnetic stirrer
    • Weigh 1.4 g thiourea under the hood with safety measures for toxic substances
      • Add 1.6 mL 37 % HCl
      • Add 2 mL distilled water
      • Add about 0.5 mL distilled water to wash the remaining thiourea out
    • Microwave
      • 250 W for 45 minutes in the microwave (no automatic temperature regulation). The temperature should be around 90 °C, if it is higher manually regulate it down.
      • 1 M NaOH for hydrolyzation, the clear solution turns cloudy (pH = 8-9)
      • 250 W for 5 minutes with no temperature regulation
    • Let it cool down to room temperature and neutralize it with HCl
    • Precipitate the solution in acetone
    • Filtrate and wash it three times with acetone
    • Dry freeze the product

    References

    (1) Chauhan, K., Singh, P., & Singhal, R. K. (2015). New Chitosan-Thiomer: An Efficient Colorimetric Sensor and Effective Sorbent for Mercury at Ultralow Concentration. ACS applied materials & interfaces, 7(47), 26069–26078. https://doi.org/10.1021/acsami.5b06078

    (2) Grosso, Roberto & De Paz, M. Violante. (2021). Thiolated-Polymer-Based Nanoparticles as an Avant-Garde Approach for Anticancer Therapies—Reviewing Thiomers from Chitosan and Hyaluronic Acid. Pharmaceutics, 13, 854. https://doi.org/10.3390/pharmaceutics13060854

    (3) Hussain Asim, M., Nazir, I., Jalil, A., Matuszczak, B., & Bernkop-Schnürch, A. (2020). Tetradeca-thiolated cyclodextrins: Highly mucoadhesive and in-situ gelling oligomers with prolonged mucosal adhesion. International journal of pharmaceutics, 577, 119040. https://doi.org/10.1016/j.ijpharm.2020.119040

    Synthesis of Cellulose Sulfate Protocol

    Materials

    • Chemicals:
      • Dry DMA (in molecular sieve)
      • Water free LiCl
      • SSP (spruce sulfite pulp from MODO PAPER)
      • NaOH (1 M)
      • SO3/Py
      • Acetone
      • 500ml Ethanol (96 % but 100 % would be best)
    • Dialysis membrane and clips
    • Syringe
    • Ultrasound bath
    • 2-neck-piston with septum
    • Cooler
    • Magnetic stirrer with implemented heater
    • Vacuum pump

    Preparation of SSP/DMA pulp

    • 10 g of SSP mashed with 1 L distilled water to a fine pulp
    • Vacuum filtrate the water out
    • Wash carefully with 500 mL ethanol and filter it (solvent exchange)
    • Wash and filter it 2-3 x with DMA
    • Fill the pulp in a glass bottle and add about 450-500 mL DMA
    • Place it overnight on a shaker
    • Filtrate the pulp and DMA mixture
    • Wash 2 x with DMA again and press as much as possible the solution out
    • Weigh the pulp and store it in a fridge
    • Warm the pulp to room temperature before use
    • Preparation of DMA/LiCl (9%)
    • 13.5 g LiCl + 150 mL DMA
    • Stirring at 60°C until completely dissolved (approx. 1 h)
    • Cool down to room temperature

    Preparation of SO3/Py in DMA

    • 6 g SO3/Py + 50 mL DMA
    • 15-20 min in ultrasound bath at 40 °C (until completely dissolved)
    • Cool down to room temperature

    Synthesis of Cellulose-Sulfate Protocol (1)

    • Dissolve 1.5 g cellulose in 150 mL DMA/LiCl (9 %)
    • Stir for 12 h at room temperature
    • Add 6 g SO3/Py in 50 mL DMA at 80 °C
      • 10 x 5 mL over 2 h
    • Stir for 1h at 80 °C
    • Cool down to room temperature
    • Precipitate in 750-1000 mL of acetone for 1 h
    • Wash 3 x with acetone and filtrate
      • Solution must be clear, otherwise filtrate again
    • Neutralize after 1 h by dissolving in 75 mL of NaOH
    • Dialyze after 1 h
        • Fill the solution in dialysis membranes and dialyze it against distilled water
      • Fill a big container with distilled water and add the full dialysis membranes
      • Put it in a sink and slightly open the distilled water so that a little amount of water drops in
    • Freeze dry when pH = 7

    References

    Muhitdinov, B., Heinze, T., Turaev, A., Koschella, A., & Normakhamatov, N., (2019). Homogenous synthesis of sodium cellulose sulfates with regulable low and high degree of substitutions with SO3/Py in N,N-dimethylacetamide/LiCl. European Polymer Journal, 119, 181-188. https://doi.org/10.1016/j.eurpolymj.2019.07.030

    Cationic, thiolated polyDADMAC-chitosan solution (own solution) Protocol

    Materials

    • Thiolated chitosan
    • PolyDADMAC high
    • 0.9 % NaCl
    • 0.1 M HCl

    Method

    • Dilute PolyDADMAC high with 0,9 % NaCl to 5 %
    • Dilute thiolated chitosan with 0.1 M HCl to 1.6 %
    • Mix the 2 solutions in a 1:4 ratio (thiolated chitosan : polyDADMAC)

    SSP capsule in PolyDADMAC Protocol

    Materials

    • Freeze dried cellulose sulfate
    • PolyDADMAC (high molecular weight, 40 %)
    • Tween 80
    • 0.9 % NaCl
    • Distilled water
    • Syringe

    Method

    Capsule solution (polyanion):

    • Dissolve 0.06 g of cellulose sulfate in 1 mL distilled water (= 6 %)
    • Vortex

    Solution (polycation):

    • Dilute 4 mL PolyDADMAC in 32 mL NaCl (0.9 %)
    • Add a small drop of Tween 80

    Drop the polyanion solution with a syringe from a height of 10-15 cm in the polycation solution.

    Disulfide-bridge-test Protocol

    Quantification of disulfide substructures (1)

    Solution preparation

    • TRIS-buffer:
      • 0.788 g TRIS-HCl in 100 mL distilled water OR
      • 121.14 g/mol Trizma-base: 0.606 g/100 mL distilled water
      • Adjust the pH to 7.6 with HCl
    • Fresh NaBH4 solution (4%):
      • 600 mg + 15 mL distilled water
    • Phosphate buffer 1 M pH = 8.0:
      • 0.95 g KH2PO4 + 15.408 g Na2HPO4 *12 H2O in 50 mL distilled water
      • Adjust pH to 8 if necessary
    • Fresh Ellman’s reagent:
      • 12 mg (5,5’-Di-thio-bis-(2-Nitro-benzoic Acid) in 3 mL Ellman’s buffer

    Sample preparation

    • Weighing 3 x 0.5 mg polymer in 15 mL falcon tubes
    • add to each 500 µL TRIS buffer and let it soak for 30 minutes
      • Chitosan: let it soak in 350 µL distilled water and after 30 minutes add 150 µL TRIS buffer

    Calibration

    • 1 mg L-cysteine-HCl in 1 mL TRIS buffer
    • Dilution series: (A = 50 µg)
      1. 100 µL stock solution + 900 µL TRIS buffer (1:10)
      2. 500 µL solution A + 500 µL TRIS buffer (1:2)
      3. 500 µL solution B + 500 µL TRIS buffer (1:2)
      4. 500 µL solution C + 500 µL TRIS buffer (1:2)
      5. 500 µL solution D + 500 µL TRIS buffer (1:2)
      6. 500 µL solution E + 500 µL TRIS buffer (1:2)
        • throw 500 µL away
      7. 500 µL TRIS buffer (blank)

    Test

    1. Add to each sample and calibration solution 1 mL fresh NaBH4 solution
    2. Incubate the samples at 37 °C for 1 h at a shaker water bath with slightly open lids (!)
    3. + 250 µL 5 M HCl (cautiously) to stop the reaction
    4. +1 mL 1 M phosphate buffer to neutralize it
    5. + 250 µL fresh Ellman’s reagent
    6. Incubate it for 90-120 min in the dark at room temperature
    7. Centrifuge the sample for 5 min at 500 rpm if the solution is not clear
    8. Measure 2 x 100 µL of the samples in a microtiter plate (absorbance: 450 nm, optimize gain)

    References

    (1) Hussain Asim, M., Nazir, I., Jalil, A., Matuszczak, B., & Bernkop-Schnürch, A. (2020). Tetradeca-thiolated cyclodextrins: Highly mucoadhesive and in-situ gelling oligomers with prolonged mucosal adhesion. International journal of pharmaceutics, 577, 119040. https://doi.org/10.1016/j.ijpharm.2020.119040

    Ellman’s Test Protocol

    Test for thiolgroups incl. Cystein (1)

    Solution preparation

    • Ellman’s buffer (Phosphatbuffer 0.5 M pH = 8.0):
      • 0.95 g KH2PO4 + 15.408 g Na2HPO4*12 H2O in 100ml distilled water
      • Adjust pH to 8
    • Fresh Ellman’s reagent
      • 3 mg (5,5-di-thio-bis-(nitro-benzoic acid) in 10ml Ellman’s buffer

    Sample preparation

    • Weighting 3x 0,5-1,0 mg polymer in 1,5 ml Eppendorf tubes
    • add to each 500 µl Ellman’s buffer and let it soak for 30 minutes

    Calibration

    • 1mg L-Cystein-HCl in 1ml Ellman’s buffer = stock solution (Cystein = 121.16 g/mol)
    • Dilution series: (A = 25 µg)
      1. 50 µl stock solution + 950 µl Ellmans buffer (1:20)
      2. 500 µl solution A + 500 µl Ellmans buffer (1:2)
      3. 500 µl solution B + 500 µl Ellmans buffer (1:2)
      4. 500 µl solution C + 500 µl Ellmans buffer (1:2)
      5. 500 µl solution D + 500 µl Ellmans buffer (1:2)
      6. 500 µl solution E + 500 µl Ellmans buffer (1:2)
        • throw 500 µl away
      7. 500 µl Ellmans’s buffer (blank)

    Test

    1. Add to each sample and calibration solution 500 µl fresh Ellman’s reagent
    2. Incubate it for 90-120 min in the dark at room temperature
    3. Centrifuge the sample for 5 min at 500 rpm
    4. Measure 2x100 µl of the samples in a microtiter plate (absorbance: 450 nm, optimize gain)

    References

    (1) Hussain Asim, M., Nazir, I., Jalil, A., Matuszczak, B., & Bernkop-Schnürch, A. (2020). Tetradeca-thiolated cyclodextrins: Highly mucoadhesive and in-situ gelling oligomers with prolonged mucosal adhesion. International journal of pharmaceutics, 577, 119040. https://doi.org/10.1016/j.ijpharm.2020.119040

    Tensile studies Protocol:

    Mucoadhesion test (1)

    Materials:

    • Porcine intestine (room temperature)
  • 90-200 mg thiolated chitosan
  • 90-200 mg unmodified chitosan
  • (1.5-3.0 mL PBS buffer [0.1 M and pH = 6,8])
  • Textur-Analysator TA.XTplus (Stable Micro Systems)

    • Measure parameters

    • Applied force: 0.100 N
    • Return distance: 20.00 mm
    • Contact time: 90 sec
    • Release force: 0.029 N

    Method

    • 30 mg of thiolated chitosan and unmodified chitosan pressed into 5.0 mm diameter flat-faced discs (triplicate or 2x triplicates)
      Pressed discs
    • Cut about 2 cm of the intestine off and slice it carefully open
      Spread out intestine
    • Carefully spread the intestine open and place it with the mucosa above on the plate without damaging it
      Texture analyzer plate
    • Close it and eventually add 200 µl PBS buffer to simulate the intestine environment
    • Attach the test discs with double sided tape (there should be no tape sticking out)
    • Rotate the plate for each test so that the undamaged part of the mucosa is used for the measuring (about 4 tests for each prepared intestine slice)

    References

    (1) Bernkop-Schnürch, A., and Steininger, S. (2000). Synthesis and Characterisation of Mucoadhesive Thiolated Polymers. International Journal of Pharmaceutics 194.2, 239-47.https://doi.org/10.1016/s0378-5173(99)00387-7

    Rheological evaluation

    Viscosity test (1)

    Materials:

    • 12 x 0.5 g porcine mucosa in 60 mm petri dish (preparation and purification are also described in the paper)
    • 200 mg thiolated chitosan
    • PBS buffer (50 mM, pH = 6.8)
    • Plate-plate rheometer (Thermo Scientific™ HAAKE MARS™, Thermo Fisher Scientific, Karlsruhe, Germany; rotor PP35Ti, D = 35 mm, gap 0.5 mm)
    Plate-plate rheometer

    Measure time

    • 0 h (triplicate)
    • 1 h (triplicate)
    • 2 h (triplicate)
    • 3 h (triplicate)

    Method

    • Incubate the mucosa at 37 °C for 30 minutes
    • Dissolve/suspend 200 mg thiolated chitosan in 10 mL PBS buffer 2 %(w/v)
    • Add 0.5 mL of the thiolated chitosan solution to the 3 petri dishes of 3 h, mix it and incubate it
    • After 1 h: add 0.5 mL of the thiolated chitosan solution to the 3 petri dishes of 2 h, mix it and incubate it
    • After another 1 h: add 0.5 mL of the thiolated chitosan solution to the 3 petri dish of 1 h, mix it and incubate it
    • After another 1 h (in total after 3 h since the first sample): add 0.5 mL of the thiolated chitosan solution to the one petri dish stir it and measure it quickly.
    • Clean the rheometer and repeat it with the other two sample of the measure time t = 0 h
    • Measure the other samples, gently scrape it off from the petri dish

    Measure parameters

    • Measured using an oscillatory strain sweep method with frequency = 1 Hz and temperature = 37 °C
    • Shear stress in a range of 0.5-500 Pa.
    • Samples equilibrated on the lower plate for 60 seconds after application until the start of the measurement

    References

    (1) Knoll, P., Le, N. N., Wibel, R., Baus, R. A., Kali, G., Asim, M. H., & Bernkop-Schnürch, A. (2021). Thiolated pectins: In vitro and ex vivo evaluation of three generations of thiomers. Acta biomaterialia, S1742-7061(21)00543-2. Advance online publication. https://doi.org/10.1016/j.actbio.2021.08.016

    Chitosan and thiolated chitosan HCl Protocol

    For water soluble chitosan hydrochloride for cytotoxicity test (1)

    Materials:

    • 1 g Chitosan or thiolated chitosan
    • 50 mL 0.1 M HCl
    • Dialysis membrane
    • Dialysis clips

    Method:

    • Solve chitosan or thiolated chitosan in 50 mL 0.1 M HCl
    • Filter the non-soluble substances
    • Fill the solution in dialysis membranes and dialyze it against distilled water
      • Fill a big container with distilled water and add the full dialysis membranes
      • Put it in a sink and slightly open the distilled water faucet so that a little amount of water drops constantly in (or change the water regularly)
    • Check the pH at the next day if it is neutralized
    • If yes, freeze dry it

      • References

      (1) Signini, R., Campana Filho, S. (1999). On the preparation and characterization of chitosan hydrochloride. Polymer Bulletin 42, 159–166. https://doi.org/10.1007/s002890050448

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