Team:IISER Bhopal/Engineering
Engineering Success
Introduction
Engineering success for our team involved constant ideation, designing, adjustment and improvement until we reached the final product - iβeta, capable of tackling diabetes by producing beta cells in vivo.
Unfortunately, our campus was locked down due to COVID-19 for essentially the entirety of our project, meaning we didn’t have access to a wet lab. Instead, we’ve designed a detailed experimental workflow, consisting of four modules of wet lab work to validate our hypothesis and demonstrate our parts’ functionality, along with a fifth module to improve our parts and correct for potential failures, and three more to build on our work and develop a device competent for testing in the clinical trial stages.
Technical Design Improvements
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Our initial plan was to express the three transcription factors on an operon, as separate ORFs. However, it was pointed out to us that we would be unable to ascertain the stoichiometric ratios of translation of our 3 factors which was for our device to operate, so we tweaked our expression system.
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We switched to a polyprotein-based BioBrick instead, where all three of our factors were joined directly by linkers under a single operon. They entered the target cell as one, achieving a roughly 1:1:1 stoichiometric ratio while being delivered to the gut cell.
In order to ensure the polyprotein was separated into 3 functional transcription factors, only in our cells of interest, we needed to find a protease specific to our target cells - crypt base columnar cells(CBCCs). -
At first, we tried to find a protease specific to our target cells - CBCCs. A promising candidate called SENP1 appeared. It helps chop SUMO tags from their conjugated proteins. A proteome-wide screen found that it was enriched in our target cells. We thought we'd found the holy grail!
But it turned out that SENP1 shared a cleavage site with multiple proteases, some of which were expressed in other cells as well. Another issue we'd overlooked was the fact that it leaves a 100 aa residue at the C-termini of our factors, which could possibly impact their function. -
Our current system does not rely on a host protease. Instead, we have sneaked in our own protease and designed it such that it is only functional in our target cells. We have used beta-catenin, the famous Wnt effector, usually degraded in most cells except the ones with an active Wnt cascade (as is present in our target cells).
We have fused a protease - Tobacco Etch Virus protease with beta-catenin which is active only in crypt cells. Our device has a multi advantage it is a protease that efficiently cleaves our transcription factors whilst maintaining cell-specificity, safety and efficacy of our system.
Following the engineering cycle of Design, Test, Improve and Research. We were able to design a
delivery system which is safe, effective and capable of producing beta cells in-vivo. To bring it to culmination we have designed several modules explaining the exact steps that would take our project from an idea to wet lab work that will give the real results.
N O T E: Unless specified, all bacterial culture work will be performed at 37°C and 200 RPM.
Extracting the plasmid (pGGA) and checking for intact restriction sites
Determining the bacterial growth kinetics
Confirming antibiotic sensitivity (Chloramphenicol, Broth + Plate)
Studying competency wrt our chosen vector
Establishing continuous cell line
(+ Checking for contaminants)
Inducing and confirming T3SS expression (SDS-PAGE)
1A. Plasmid Extraction
We plan to assemble our parts onto the Golden Gate destination vector pGGA, shown here. First, we will transform NEB5α competent E. coli cells with the lyophilised vector, and extract it with a standard miniprep kit.Then, we will confirm linearisation by restriction digestion with BsaI-HF, followed by visualisation on an agarose gel.
The plasmid pGGA will be extracted (after stock revival, using LB-Cm plates) from its DH5α stock using the QIAGEN Miniprep protocol, described here.
The plasmid’s integrity will be checked by digesting 5 μg of the extracted plasmid with BsaI in the recommended buffer at 37OC for 3 hours, followed by resolution on a 1% agarose gel. In parallel, a mock-digested plasmid will also be resolved.
Protocol (In Brief)
- Culturing plasmid stock in LB-Cm broth to mid-log phase.
- Pelleting cells down, resuspending in DNA-protective buffer.
- Alkaline cell lysis, followed by renaturation in acetate buffer.
- Supernatant extraction.
- Plasmid purification via silica spin column.
This will allow us to check the plasmid’s size as well as the presence of BsaI restriction sites, thereby refining our troubleshooting protocols by eliminating a potential source of error. The extracted plasmid also allows us to test our chassis’ competency.
Troubleshooting
Detailed protocol
Analyzing fractions saved from each step in the procedure on an agarose gel to
find the source of error.
Errors
- Presence of RNA in the eluate : Adding RNase A to the resuspension buffer.
- Presence of genomic DNA in the eluate: Handling the lysate gently after addition of lysis buffer to prevent shearing and Reducing the time for lysis reaction (Maximum 5 mins).
- Little or no DNA in eluate: Checking the pH of the elution buffer (desired pH 8.5).
1B. Growth Kinetics Measurement
We will measure the growth kinetics of our strains of interest, in order to finetune our protocols as per the concentrations recommended.
The growth kinetics of E. coli K-12, SIEC and SIEC-eLEE5 will be studied, both in the presence and absence of IPTG and L-arabinose, in order to attempt to observe any inducer-dependent or insert-dependent effects on observed growth.
The experiment will be conducted in the LB medium, using the protocol described here.
Protocol (In Brief)
- Inoculating freshly grown bacterial culture in 150 ml of nutrient media with different IPTG and L-arabinose concentrations: 0, 0.5, 1, 2, 3 and 4 g/L.
- Collecting 2 ml of samples from each flask at following time intervals: 0 hr, 4 hrs, 6hrs, 8hrs, 10 hrs, 12hrs, 14 hrs, 16 hrs, 18hrs, 20 hrs, 22 hrs and 24 hrs.
- Collecting 2 ml of samples from each flask at following time intervals: 0 hr, 4 hrs, 6hrs, 8hrs, 10 hrs, 12hrs, 14 hrs, 16 hrs, 18hrs, 20 hrs, 22 hrs and 24 hrs.
1C .Antibiotic Sensitivity Assay
We will confirm that our strain is sensitive to the antibiotic chloramphenicol at 35 μg/ml, in order to ensure selection for optimal plasmid stability.
Protocol (In Brief):
- E. coli K-12, SIEC and SIEC-eLEE5 will be cultured in LB for 12-16 hours, before a 1:100 dilution into LB-Cm. In parallel, 20 μL of the saturated primary culture will be streaked onto an LB-Cm plate.
- Growth will be assayed using spectrophotometry after 8 hours (for the broth culture), and by looking for colonies on the plate after O/N incubation.
- Cultures in plain LB broth and plates will be used as controls, in order to determine chloramphenicol-dependent effects on growth.
Troubleshooting: Preparing fresh stock of chloramphenicol.
- Adding 2.72g chloramphenicol to a Pyrex bottle containing 80mL 100% EtOH.
- Adding the magnetic stir bar into the solution and placing the Pyrex bottle on the magnetic stirrer set at 300-600 rpm.
- Aliquoting (1mL) into microcentrifuge tubes.
1D. Competency Check
Competency in our strains will be chemically induced, and the strains will be transformed with pGGA using the protocol described here.
In parallel, they will be transformed with pUC19, as a positive control.
Protocol (In Brief)
- Subculturing overnight culture to 0.4 OD.
- Conducting a CaCl2 wash.
- Transforming the competent cells via heat shock.
- Suspending transformed cells in SOC, incubating for 3 hrs.
- Spread plating the 1:10, 1:1000 and 1:100000 dilutions of the cells on selective LB plates.
This will give us competent cell stocks for use in future modules, allowing us to troubleshoot our cloning procedures in the future.
Troubleshooting: Extracting the pGGA plasmid again. Refer to Module-1 Protocol-A.
1E. Human Cell Line Maintenance
We will establish our continuous lines with the protocol described here
Protocol (In Brief):
Complete growth medium- OptiMEM 1 Reduced Serum Medium, 20 mM HEPES, 10 mM GlutaMAX, 10 ng/mL Epidermal Growth Factor (EGF) and fetal bovine serum (FBS) to a final concentration of 4%.
- Removing and discarding the culture medium.
- Briefly rinsing the cell layer with free Dulbecco's phosphate-buffered saline (D-PBS) for removal of all traces of serum containing trypsin inhibitor.
- Adding Trypsin-EDTA solution to the flask.
- Observing the cells under an inverted microscope (checking the dispersal of the cell layer).
- Note: Avoid agitating the cells while waiting for them to detach to avoid clumping.
- Adding 6.0 to 8.0 mL of complete growth medium and aspirating the cells by gentle pipetting.
- Adding appropriate aliquots of the cell suspension to new culture vessels.
- Incubating cultures at 37°C.
Troubleshooting:
Checking for suspended contaminants through DAPI staining and microscopy with the protocol described here.
- Removing the cell culture medium and incubating the cells for 15 minutes with 4% PFA.
- Washing the cells twice with 1X PBS.
- Incubating cells with DAPI solution for about a minute.
- Washing the cells twice or thrice with 1X PBS.
- Keeping the washed cells in PBS.
- Observing the cells under an epifluorescent microscope.
1F. T3SS Validation
The expression of the T3SS will be induced and validated with the protocol described here. This will be performed with SIEC and SIEC-eLEE5, with E. coli K-12 as a negative control. Performing this experiment allows us to check for the presence of our delivery system, making it easier to troubleshoot certain experiments in Modules 3, 4 and 5.
Protocol (In Brief):
- Preparation of early-log phase secondary culture.
- Aliquoting, preserving uninduced control.
- Pelleting sample, resuspending in IPTG-supplemented media, incubation.
- Pelleting induced sample post-incubation, preparation, SDS-PAGE.
Troubleshooting:
- Checking the survival of K-12 in IPTG-supplemented media.
- Checking the growth curves of SIEC and SIEC-eLEE5, and E. coli K-12 in IPTG-supplemented media.
Obtaining and amplifying synthetically generated construct
Loading construct onto vector (pGGA) using Golden Gate assembly
Transformation into cloning host + Screening
Transformation into test and control host (E. coli K-12)
Inducing and confirming effector expression (SDS-PAGE)
2A. Obtaining and amplifying the Constructs
Our main biobrick would consist of the following:
It would contain an L-arabinose-inducible expression system controlling a gene for a polyprotein containing the three factors of interest (each with its own C-terminal NLS), along with an N-terminal T3SS targeting signal called Map20. The three proteins will be separated by recognition sites (labelled TEVs) for the Tobacco Etch Virus Protease (TEVp). Each factor would also be FLAG-tagged, for experimental purposes.
In a separate transcriptional unit, it would also contain the β-Catenin-TEVp fusion that constitutes one of our targeting systems.
We will construct this biobrick synthetically, and amplify it with PCR, adding in the BsaI restriction sites and the overhangs required.
The cassettes of interest will be procured by chemical synthesis from IDT. The lyophilised samples obtained will be suspended in the appropriate amount of nuclease-free water (NFW), and amplified using PCR, following the protocol described here.
Protocol (In Brief):
- Assembling and thawing all the required reagents (Q5 High-Fidelity 2X Master Mix, 10 µM Forward Primer, 10 µM Reverse Primer, Template DNA and Nuclease-Free Water) on ice.
- Mixing appropriate amounts of the reaction components in a PCR tube.
- Transferring the reactions to a thermocycler preheated to the denaturation temperature (98°C).
- 35 cycles of Denaturation, Annealing (Tm-5) and Extension followed by a final extension.
The DNA thus obtained will be purified and extracted from a 1% agarose gel (to remove primers and incomplete amplification products) using the protocol described here.
Protocol (In Brief):
- Dissolving cut gel slices in a buffer containing a pH indicator for optimal dissolution.
- Applying the mixture to the QIAquick spin column.
- Washing away the impurities and eluting pure DNA via a small volume of NFW.
This gets us the material we need to carry out our cloning work.
Troubleshooting: PCR troubleshooting guide
- We will rerun the reaction after increasing or decreasing the number of cycles.
- We will rerun the reaction after increasing or decreasing the concentration of primers.
- We will rerun the reaction after increasing or decreasing the concentration of the template DNA.
- We will rerun the reaction after checking the primers for self-complementarity and cross-complementarity.
- We will rerun the reaction after checking for additional complementary regions for the primers within the template DNA.
- We will rerun the reaction after increasing or decreasing the length of the primers.
- We will rerun the reaction after increasing or decreasing the annealing temperature.
- We will rerun the reaction after recalculating the primer Tm values.
2B. Golden Gate Assembly
Our parts will be assembled onto our backbone using the single-pot digestion-ligation reaction described here.
Protocol (in brief):
- Adding inserts and intact backbone into a single reaction mixture, and adding BsaI-HFv2, T4 DNA Ligase and the appropriate ATP-supplemented buffer.
- Running the reaction at 37OC for 1 hr.
- Incubating at 60OC for 5 min.
2C. Transformation and Screening
The ligated product will first be transformed into DH5α using the protocol described here.
Insert release will be validated following plasmid extraction and a double digest with BamHI-HF and XhoI in the CutSmart buffer at 37OC for 3 hours. The bands derived thusly will be resolved and visualised on a 1% agarose gel.
Protocol (In Brief):
- Subculturing overnight culture to 0.4 OD.
- Conducting a CaCl2 wash.
- Transforming the competent cells via heat shock.
- Suspending transformed cells in SOC, incubating for 3 hrs.
- Spread plating the 1:10, 1:1000 and 1:100000 dilutions of the cells on LB-Cm plates.
The inserts will then be sequenced using the protocols described here.
Brief protocol for preparing the reaction mixture
- Adding the template DNA to the reaction mastermix containing four nucleotides (dATP, dTTP, dCTP, dGTP) and the labelled ddNTPs (ddATP, ddTTP, ddCTP, ddGTP).
- Adding the primer and DNA polymerase.
The screening procedures described above will allow us to verify the integrity of our inserts, streamlining our future troubleshooting work.
Troubleshooting: We will prepare a fresh stock of chloramphenicol with the protocol described here.
Protocol (In Brief):
Stock concentration: 25mg/mL in 100% EtOH
- Adding 2.72g chloramphenicol to a Pyrex bottle containing 80mL 100% EtOH.
- Adding the magnetic stir bar into the solution and placing the Pyrex bottle on the magnetic stirrer set at 300-600 rpm.
- Aliquoting (1mL) into microcentrifuge tubes.
2D. Transformation into Test Hosts
The plasmid will be extracted and transformed into competent E. coli K-12, SIEC and SIEC-eLEE5 using the protocol described here.
Protocol (In Brief):
- Subculturing overnight culture to 0.4 OD.
- Conducting a CaCl2 wash.
- Transforming the competent cells via heat shock.
- Suspending transformed cells in SOC, incubating for 3 hrs.
- Spread plating the 1:10, 1:1000 and 1:100000 dilutions of the cells on LB-Cm plates.
Troubleshooting:
We will analyze the fractions saved from each step in the procedure on an agarose gel to find the source of error with the protocol described here.
Errors
2. Presence of genomic DNA in the eluate: We will handle the lysate gently after addition of the lysis buffer to prevent shearing and reduce the time for lysis reaction (Maximum 5 mins).
3. Little or no DNA in eluate: We will check the pH of the elution buffer (optimum pH 8.5).
2E. Expression and Validation
The expression of the desired factors will be induced with L-arabinose and observed using the protocol described here. This will be performed in K-12, SIEC and SIEC-eLEE5 in the presence or absence of IPTG (to induce the expression of the T3SS).
Protocol (In Brief):
- Preparation of early-log phase secondary culture.
- Aliquoting, preserving uninduced control.
- Pelleting sample, resuspending in L-arabinose-supplemented media, incubation.
- Pelleting induced sample post-incubation, preparation, SDS-PAGE.
Performing this experiment allows us to check for the expression of our devices, making it easier to troubleshoot certain experiments in Modules 3, 4 and 5.
Troubleshooting:
- We will check the survival of K-12 in IPTG-supplemented media.
- We will check the growth curves of SIEC and SIEC-eLEE5, and E. coli K-12 in IPTG-supplemented media.
- If the SDS-PAGE result yields bands corresponding to the individual factors, it is likely to be the result of TEVp activity. We will attempt to correct this by coexpressing the T3SS-associated chaperone CesT (developed by iGEM Copenhagen 2018) on a compatible vector, to sequester the protease.
Observing T3SS secretion into media (SDS-PAGE)
Visualising and quantifying binding and intimate contact (Microscopy + Bound Cell Counting)
Observing effector translocation and nuclear entry (Fractionation + Western Blotting)
Quantifying insulin output (ELISA)
3A. Observing T3SS secretion into media
We will assay T3SS secretion downstream of expression by inducing secretion into the culture
medium and observing the secreted products.
We will observe the secretion of our effectors using the protocol described here
This will be performed on L-arabinose and IPTG-induced K-12, SIEC and SIEC-eLEE5 harbouring
the recombinant plasmid, with controls lacking each (and both) of the inducers.
Protocol (In Brief):
- Inducing the expression of the relevant parts (See Module 1).
- Centrifugation and supernatant collection.
- Protein extraction and TCA-precipitation.
- Visualisation through SDS-PAGE.
To troubleshoot this, we will:
- Use Tir, EspA, B and D as positive controls. If these give positives, but the effector gives negatives, the construct is faulty. We will attempt to rectify this by altering our leader.
- If the positive controls give negatives, we will add EDTA to our media to attempt further induction.
3B. Visualising and Quantifying Binding and Intimate Contact
We will visualise the T3SS-dependent binding of our bacteria to HIEC-6 using the protocol described here. This will be performed using SIEC and SIEC-eLEE5, either in the presence or absence of IPTG, in order to demonstrate dependence on the T3SS, as well as the Tir-intimin system.
Protocol (In Brief):
- Growing SIEC-eLEE5 bacteria in LB with IPTG to mid log phase, and incubating with HIEC-6 cells with and without IPTG for 3 or 6 hours.
- Washing, fixing, and staining the samples for fluorescence microscopy to visualise bacteria (DAPI), F-actin (phalloidin), and nuclei (DAPI).
Additionally, we will quantify this attachment by counting the number of bacteria attached to each human cell using the attached protocol.
Protocol (In Brief):
- Inoculating bacteria at 1:50 dilution in 24-well microplates containing incomplete HIEC-6 cell monolayers in DMEM with 15 mM HEPES, 2% FBS and 2% D-mannose (to block type I fimbriae-mediated adherence).
- Washing with phosphate buffered saline (PBS) after 3 hours of incubation.
- Incubating for another 3 hours after adding fresh medium (6-h assay).
- Fixing the preparations with methanol at room temperature for 30 min after several washes.
- Staining with May-Grünwald/Giemsa and analyzing under immersion light microscopy.
- Lysing of HIEC-6 cells in sterile distilled water (30 min at 37°C) after 6 hours of incubation.
- Serially diluting and spreading of the bacterial suspensions on MacConkey agar for colony forming units/mL (CFU/mL) determination.
This allows us to determine that the T3SS is indeed functional, and can serve as a competent adhesin, in addition to being able to secrete factors into the cytosol of intestinal epithelial cells.
Troubleshooting:
- We will ensure that the system can sense microfilament perturbations by observing
cytoskeletal shifts after Cytochalasin D introduction using phalloidin.
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We will independently observe HIEC-6 and our chassis under an epifluorescence
microscope using DAPI staining, to ensure that our system can detect those signals.
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If the problem isn't resolved, we will attempt to replicate these results in
HeLa, to control for cell-specific outcomes.
- We will ensure that the system can sense microfilament perturbations by observing cytoskeletal shifts after Cytochalasin D introduction using phalloidin.
- We will independently observe HIEC-6 and our chassis under an epifluorescence microscope using DAPI staining, to ensure that our system can detect those signals.
- If the problem isn't resolved, we will attempt to replicate these results in HeLa, to control for cell-specific outcomes.
3C. Observing Effector Translocation, Proteolysis and Nuclear Entry
We will observe translocation, proteolysis and nuclear entry downstream of T3SS binding using the protocol described here . The antibodies used in the Western blotting work will be directed at FLAG tags conjugated with each of the transcription factors.
The proteolytic event can be observed using size determination on the Western blot, and the subcellular localisation of the factors can be observed with Western blots against various cellular fractions (cytoplasmic or nuclear).
Protocol (Extracting nuclear fraction)
- Pre Incubating and washing the cells with PBS.
- Dislodging and pelleting the cells via centrifugation.
- Resuspending the pellet in the cell lysis buffer and incubating on ice for 15‐20 min with intermittent mixing.
- Vortexing the tubes to disrupt cell membranes and then centrifuging them (12,000 g at 4°C) for 10 min.
- Storing the supernatant (cytoplasmic extract) at ‐70°C. Resuspending the pelleted nuclei in the nuclear extraction buffer and incubating on ice for 30 min.
- Washing the pelleted nuclei thrice with the cell lysis buffer.
- Resuspending the pelleted nuclei in the nuclear extraction buffer and incubating on ice for 30 min.
- Collecting the nuclear extract by centrifugation (12,000 g at 4°C) for 15 min.
- Storing the nuclear fraction at ‐70°C.
Protocol (in brief, following fractionation):
- Separating Purified proteins (PDX1, MAFA and NGN3) from the nuclear fraction by SDS-PAGE and transferring them to PVDF membranes.
- Pre Incubating PDX1, MAFA and NGN3 with 2 mM PMSF to avoid proteolysis.
- Adsorbing PDX1, MAFA and NGN3 on nitrocellulose membranes.
- Blocking the membranes from the blot and dot blot assays overnight and incubating with PDX1 at 5 g/ml in binding buffer for 1.5 hours
- Incubating with mouse anti-FLAG antibody for 1 hour followed by goat anti-mouse IgG secondary antibody, and horseradish peroxidase conjugated for 1 hour.
- Detecting the reaction via a western chemiluminescent HRP substrate kit.
This allows us to verify that our effectors are cleaved in the cytosol, and that they enter the nucleus of our target cells post infection, in a T3SS-dependent manner.
Troubleshooting:
- If our loading controls show up negative, we will incorporate more potent protease inhibitors into our extraction protocol, and follow standard troubleshooting procedures for fractionation.
- We will stain the nuclear and cytoplasmic fractions with antibodies against cytoplasmic and nuclear markers, respectively, to check for cross-fraction contamination.
3D. Quantifying Insulin Output
We will quantify insulin secretion using Insulin-ELISA from the culture supernatant. The relevant protocols are described here.This will be done at different ambient glucose levels, to demonstrate that our system is glucose-responsive.
Protocol (In Brief):
- Introducing factors into cells (See Above).
- Extracting supernatant at various time points (0-72 hrs).
- Adding samples to wells, incubating as prescribed.
- Removing unbound fluid, washing.hrs.
- Adding antibodies and reagents as recommended.
- Stopping reaction, measuring OD450, quantifying against standard.
Troubleshooting:
- We will use the supplied standard to verify whether the detection kit is functional.
- We will run Western blots from the supernatant against insulin in order to qualitatively verify its presence.
Constructing and validating antibody display apparatus
(See Module 2)
Studying the impact of antibody display on binding dynamics
(See Module 3B)
Constructing and validating expression of kill switch
(See Module 2)
Studying kill switch functionality
4A. Antibody Display
To target our chassis towards crypt base columnar cells (CBCCs), we will display the variable heavy chain of a single-domain antibody (sdAb) on the bacterial surface, targeted at moieties specific to CBCCs. We have chosen to target the cell-surface GPCR and noted CBCC marker Lgr5.
We will construct this device using the techniques introduced in Module 2, and test it using the techniques introduced in Module 3B.
A1:Obtaining and amplifying the Constructs
The cassettes of interest will be procured by chemical synthesis from IDT. The lyophilised samples obtained will be suspended in the appropriate amount of nuclease-free water (NFW), and amplified using PCR, following the protocol described here.
Protocol (In Brief):
- Assembling all reaction components Q5 High-Fidelity 2X Master Mix, 10 µM Forward Primer 10 µM Reverse Primer, Template DNA and Nuclease-Free Water on ice.
- Mixing the reaction components in a PCR tube.
- Transferring the reactions to a thermocycler preheated to the denaturation temperature (98°C).
- 35 cycles of Denaturation, Annealing (Tm-5) and Extension at the temperature followed by final extension.
The DNA thus obtained will be purified and extracted from a gel (to remove primers and incomplete amplification products) using the protocol described here.
Protocol (In Brief):
- Dissolving the gel slices in a buffer containing a pH indicator, for optimal binding.
- Applying the mixture to the QIAquick spin column.
- Washing away the impurities and eluting pure DNA via a small volume of NFW.
A2:Golden Gate Assembly
Our parts will be assembled onto our backbone using the single-pot digestion-ligation reaction described here.
Protocol (In Brief):
- Adding inserts and intact backbone into a single reaction mixture, and adding BsaI-HFv2, T4 DNA Ligase and the appropriate ATP-supplemented buffer.
- Running the reaction at 37OC for 1 hr.
- Incubating at 60OC for 5 min.
A3:Transformation and Screening
The ligated product will first be transformed into DH5α using the protocol described here.
Protocol (In Brief):
- Subculturing overnight culture until OD reaches 0.4
- CaCl2 wash
- Transforming the competent cells via heat shock method
- Spread plating the 1:10, 1:1000 and 1:100000 dilutions of the cells on selection plates (Chloramphenicol resistant).
Insert release will be validated following plasmid extraction and a double digest with BamHI-HF and XhoI in the CutSmart buffer at 37OC for 3 hours. The bands derived thusly will be resolved and visualised on a 1% agarose gel.
Protocol (In Brief):
- Culturing plasmid stock in LB-Cm broth to mid-log phase.
- Pelleting cells down, resuspending in DNA-protective buffer.
- Alkaline cell lysis, followed by renaturation in acetate buffer.
- Supernatant extraction.
- Plasmid purification via silicate spin column.
The inserts will then be sequenced using the protocols described here.
A4:Transformation into Test Hosts
The plasmid will be extracted and transformed into competent E. coli K-12, SIEC and SIEC-eLEE5 using the protocols described above.
Protocol (In Brief):
- Subculturing overnight culture until OD reaches 0.4
- CaCl2 wash
- Transforming the competent cells via heat shock method
- Spread plating the 1:10, 1:1000 and 1:100000 dilutions of the cells on selection plates (Chloramphenicol resistant)
4B. Antibody Display Dynamics
We will visualise the antibody mediated T3SS-dependent binding of our bacteria to HIEC-6 using the protocol described here. This will be performed using SIEC and SIEC-eLEE5, either in the presence or absence of IPTG, in order to demonstrate dependence on the T3SS, as well as the Tir-intimin system.
Protocol (In Brief):
- SIEC-eLEE5 bacteria will be grown in LB with IPTG to mid log phase, and incubated with HIEC-6 cells with and without IPTG for 3 and 6 hours respectively.
- The samples will be washed after infection, fixed, and stained for fluorescence microscopy to visualize bacteria (DAPI), F-actin (phalloidin), and nuclei (DAPI).
Additionally, we will quantify this attachment by counting the number of bacteria attached to each human cell using the attached protocol.
Protocol (In Brief):
- Inoculating bacteria at 1:50 dilution in 24-well microplates containing incomplete HIEC-6 cell monolayers in DMEM with 15 mM HEPES, 2% FBS and 2% D-mannose (to block type I fimbriae-mediated adherence).
- Washing with phosphate buffered saline (PBS) after 3 h of incubation at 37°C.
- Incubating for another 3 h after adding fresh medium (6-h assay).
- Fixing the preparations with methanol at room temperature for 30 min after several washes.
- Staining with May-Grünwald/Giemsa (Merck, NJ, United States) and analyzing under immersion light microscopy.
4C. Constructing and validating expression of kill switch
Our kill switch integrates inputs from a phosphate-sensitive circuit in the pho regulon, and
feeds it into the E2-IM2 toxin-antitoxin system via the repression of IM2, mediated by the the
bacteriophage lambda cI repressor expressed under PhoB’s promoter.
We will construct this BioBrick in E. coli K-12, and test it in the manner described below.
C1: Obtaining and amplifying the Constructs
The cassettes of interest will be procured by chemical synthesis from IDT. The lyophilised samples obtained will be suspended in the appropriate amount of nuclease-free water (NFW), and amplified using PCR, following the protocol described here.
Protocol (In Brief):
- Dissolving the gel slices in a buffer containing a pH indicator, for optimal binding.
- Applying the mixture to the QIAquick spin column.
- Washing away the impurities and eluting pure DNA via a small volume of NFW.
The DNA thus obtained will be purified and extracted from a gel (to remove primers and incomplete amplification products) using the protocol described here.
Protocol (In Brief):
- Dissolving the gel slices in a buffer containing a pH indicator, for optimal binding.
- Applying the mixture to the QIAquick spin column.
- Washing away the impurities and eluting pure DNA via a small volume of NFW.
C2: Golden Gate Assembly
Our parts will be assembled onto our backbone using the single-pot digestion-ligation reaction described here.
Protocol (In Brief):
- Adding inserts and intact backbone into a single reaction mixture, and adding BsaI-HFv2, T4 DNA Ligase and the appropriate ATP-supplemented buffer.
- Running the reaction at 37OC for 1 hr.
- Incubating at 60°C for 5 min.
C3: Transformation and Screening
The ligated product will first be transformed into DH5α using the protocol described here.
Protocol (In Brief):
- Subculturing overnight culture until OD reaches 0.4
- CaCl2 wash
- Transforming the competent cells via heat shock method
- Spread plating the 1:10, 1:1000 and 1:100000 dilutions of the cells on selection plates (Chloramphenicol resistant).
Insert release will be validated following plasmid extraction and a double digest with BamHI-HF and XhoI in the CutSmart buffer at 37OC for 3 hours. The bands derived thusly will be resolved and visualised on a 1% agarose gel.
Protocol (In Brief):
- Culturing plasmid stock in LB-Cm broth to mid-log phase.
- Pelleting cells down, resuspending in DNA-protective buffer.
- Alkaline cell lysis, followed by renaturation in acetate buffer.
- Supernatant extraction.
- Plasmid purification via silicate spin column.
The ligated product will first be transformed into DH5α using the protocol described here. The inserts will then be sequenced using the protocols described here.
C4: Transformation into Test Hosts
The plasmid will be extracted and transformed into competent E. coli K-12, SIEC and SIEC-eLEE5 using the protocols described above.
Protocol (In Brief):
- Subculturing overnight culture until OD reaches 0.4.
- CaCl2 wash
- Transforming the competent cells via heat shock method
- Spread plating the 1:10, 1:1000 and 1:100000 dilutions of the cells on selection plates (Chloramphenicol resistant)
The expression of the desired factors will be induced with L-arabinose and observed using the protocol described here. This will be performed in K-12, SIEC and SIEC-eLEE5 in the presence or absence of IPTG (to induce the expression of the T3SS).
Protocol (In Brief):
- Preparation of early-log phase secondary culture.
- Aliquoting, preserving uninduced control.
- Pelleting sample, resuspending in IPTG-supplemented media, incubation.
- Pelleting induced sample post-incubation, preparation, SDS-PAGE.
4D. Studying the Kill Switch
We will prepare our strains as described here, and obtain cell counts and viable cell counts at various time points post induction using spectrophotometry (by measuring the OD600 values of 1 mL aliquots) and CFU analysis (by spreading serially diluted aliquots on LB-Cm plates), respectively.
Protocol (In Brief):
- Isolation of early log phase cells.
- Resuspension in MOPS, dilution into phosphate-controlled solution.
- Aliquoting at various time points, OD measurement.
- Serial dilution, spread plating, O/N incubation.
- Colony counting.
Constructing and validating secretion-tagged reporters (See Module 2)
Studying the impact of improved linker on expression, folding and secretion
Constructing and validating effector with improved protease sensitivity (See Module 2)
Observing improved protease sensitivity
5A. Constructing and Validating Secretion-Tagged Reporters
During our survey of the iGEM repository, we noticed that the T3SS secretion signal we have chosen (Map20), appears to inhibit either protein expression or folding in a chaperone-independent manner. We posit that this is the result of folding interference between Map20 and the protein it is fused to, raising concerns about the fusion’s ability to fold correctly in the target cells. We intend to rectify this issue by introducing a long, flexible linker to separate the two domains.
We will build Map20 - Linker - mCherry - β-lactamase and Map20 - mCherry - β-lactamase constructs to report expression and folding, both in our chassis (with mCherry) and in human cells it is translocated into (with β-lactamase). We will FLAG-tag these constructs to verify their presence, for troubleshooting purposes. Additionally, we will use GFP for normalisation.
The cassettes of interest will be procured by chemical synthesis from IDT. The lyophilised samples obtained will be suspended in the appropriate amount of nuclease-free water (NFW), and amplified using PCR, following the protocol described here.
Protocol (In Brief):
- Assembling and thawing all the required reagents (Q5 High-Fidelity 2X Master Mix, 10 µM Forward Primer, 10 µM Reverse Primer, Template DNA and Nuclease-Free Water) on ice.
- Mixing appropriate amounts of the reaction components in a PCR tube.
- Transferring the reactions to a thermocycler preheated to the denaturation temperature (98°C).
- 35 cycles of Denaturation, Annealing (Tm-5) and Extension followed by a final extension.
The DNA thus obtained will be purified and extracted from a 1% agarose gel (to remove primers and incomplete amplification products) using the protocol described here.
Protocol (In Brief):
- Dissolving cut gel slices in a buffer containing a pH indicator for optimal dissolution.
- Applying the mixture to the QIAquick spin column.
- Washing away the impurities and eluting pure DNA via a small volume of NFW.
This gets us the material we need to carry out our cloning work.
Troubleshooting:
- Rerunning the reaction after increasing or decreasing the number of cycles.
- Rerunning the reaction after increasing or decreasing the concentration of primers.
- Rerunning the reaction after increasing or decreasing the concentration of the template DNA.
- Checking the primers for self-complementarity and cross-complementarity.
- Checking for additional complementary regions for primers within the template DNA.
- Rerunning the reaction after increasing or decreasing the length of the primers.
- Rerunning the reaction after increasing or decreasing the annealing temperature.
- Rerunning the reaction after recalculating the primer Tm values.
A1. Golden Gate Assembly
Our parts will be assembled onto our backbone using the single-pot digestion-ligation reaction described here.
Protocol (in brief):
- Adding inserts and intact backbone into a single reaction mixture, and adding BsaI-HFv2, T4 DNA Ligase and the appropriate ATP-supplemented buffer.
- Running the reaction at 37OC for 1 hr.
- Incubating at 60OC for 5 min.
A2. Transformation and Screening
The ligated product will first be transformed into DH5α using the protocol described here.
Insert release will be validated following plasmid extraction and a double digest with BamHI-HF and XhoI in the CutSmart buffer at 37OC for 3 hours. The bands derived thusly will be resolved and visualised on a 1% agarose gel.
Protocol (In Brief):
- Subculturing overnight culture to 0.4 OD.
- Conducting a CaCl2 wash.
- Transforming the competent cells via heat shock.
- Suspending transformed cells in SOC, incubating for 3 hrs.
- Spread plating the 1:10, 1:1000 and 1:100000 dilutions of the cells on LB-Cm plates.
The inserts will then be sequenced using the protocols described here.
Brief protocol for preparing the reaction mixture
- Adding the template DNA to the reaction mastermix containing four nucleotides (dATP, dTTP, dCTP, dGTP) and the labelled ddNTPs (ddATP, ddTTP, ddCTP, ddGTP).
- Adding the primer and DNA polymerase.
The screening procedures described above will allow us to verify the integrity of our inserts, streamlining our future troubleshooting work.
Troubleshooting:
Preparing fresh stock of chloramphenicol. Protocol
Protocol (in brief):
Stock concentration: 25mg/mL in 100% EtOH
- Adding 2.72g chloramphenicol to a Pyrex bottle containing 80mL 100% EtOH.
- Adding the magnetic stir bar into the solution and placing the Pyrex bottle on the magnetic stirrer set at 300-600 rpm.
- Aliquoting (1mL) into microcentrifuge tubes.
A3. Transformation into Test Hosts:
The plasmid will be extracted and transformed into competent E. coli K-12, SIEC and SIEC-eLEE5 using the protocol described here.
Protocol (In Brief):
- Subculturing overnight culture to 0.4 OD.
- Conducting a CaCl2 wash.
- Transforming the competent cells via heat shock.
- Suspending transformed cells in SOC, incubating for 3 hrs.
- Spread plating the 1:10, 1:1000 and 1:100000 dilutions of the cells on LB-Cm plates.
Troubleshooting:
Detailed protocol
- Analyzing fractions saved from each step in the procedure on an agarose gel to find the source of error.
Errors:
- Presence of RNA in the eluate : Adding RNase A to the resuspension buffer.
- Presence of genomic DNA in the eluate: Handling the lysate gently after addition of lysis buffer to prevent shearing and Reducing the time for lysis reaction (Maximum 5 mins).
- Little or no DNA in eluate: Checking the pH of the elution buffer (desired pH 8.5) and
A4. Expression and Validation
The expression of the desired factors will be induced with L-arabinose and observed using the protocol described here. This will be performed in K-12, SIEC and SIEC-eLEE5 in the presence or absence of IPTG (to induce the expression of the T3SS).
Protocol (In Brief):
- Preparation of early-log phase secondary culture.
- Aliquoting, preserving uninduced control.
- Pelleting sample, resuspending in L-arabinose-supplemented media, incubation.
- Pelleting induced sample post-incubation, preparation, SDS-PAGE.
Performing this experiment allows us to check for the expression of our devices, making it easier to troubleshoot certain experiments in Modules 3, 4 and 5.
Troubleshooting:
- Checking the survival of K-12 in IPTG-supplemented media.
- Checking the growth curves of SIEC and SIEC-eLEE5, and E. coli K-12 in IPTG-supplemented media.
Protocol: http://parts.igem.org/Part:BBa_K2871000
5B. Testing the Secretion Tag
mCherry will be used to report folding in the chassis, and β-lactamase will be used to report folding in target cells. Together, this dual reporter system allows us to study the protein’s ability to fold across different contexts.
Protocol (In Brief):
- Induce expression with L-arabinose (See Module 1).
- Pellet down induced cells, resuspend in LB-Cm.
- Measure mCherry fluorescence using spectrometric quantification (Excitation at 587 nm, Emission at 610 nm).
- Measure GFP fluorescence using spectrometric quantification (Excitation at 395 nm, Emission at 509 nm).
- Induce T3SS and effector expression (See Module 1).
- Translocate effector into HIEC-6 (See Module 3).
- Wash with HBSS.
- Add HBSS + CCF2/AM (Substrate Solution).
- Incubate at room temperature for 90 min.
- Observe β-lactamase fluorescence on plate reader (Excitation at 405 nm, Emission at 460 and 530 nm).
This assay helps inform our choice of leader, and validates our improvement.
Troubleshooting:
- We will run Western blots against FLAG in order to verify the protein's presence in both the soluble and insoluble fraction.
- If the fusion is insoluble, we will attempt to express the T3SS-associated chaperone CesT to correct it.
5C.Constructing and validating effector with improved protease sensitivity (See Module 2)
Protease Cleavage Site
We intend to explore replacing the TEVs cleavage site with the 2x TEVs site, containing a pair of TEVp cleavage sites. It has long been thought that such a site would be more sensitive to TEVp than the conventional single cleavage site, although this has not been empirically verified.
We will test this improved part in HIEC-6 (after cloning into SIEC-eLEE5) using the FlipGFP protease cleavage reporter, with mCherry for normalisation.
The cassettes of interest will be procured by chemical synthesis from IDT. The lyophilised samples obtained will be suspended in the appropriate amount of nuclease-free water (NFW), and amplified using PCR, following the protocol described here.
Protocol (In Brief):
- Assembling and thawing all the required reagents (Q5 High-Fidelity 2X Master Mix, 10 µM Forward Primer, 10 µM Reverse Primer, Template DNA and Nuclease-Free Water) on ice.li>
- Mixing appropriate amounts of the reaction components in a PCR tube.
- Transferring the reactions to a thermocycler preheated to the denaturation temperature (98°C).li>
- 35 cycles of Denaturation, Annealing (Tm-5) and Extension followed by a final extension.
The DNA thus obtained will be purified and extracted from a 1% agarose gel (to remove primers and incomplete amplification products) using the protocol described here.
Protocol (in brief):
- Dissolving cut gel slices in a buffer containing a pH indicator for optimal dissolution.
- Applying the mixture to the QIAquick spin column.
- Washing away the impurities and eluting pure DNA via a small volume of NFW.
This gets us the material we need to carry out our cloning work.
Troubleshooting:
- Rerunning the reaction after increasing or decreasing the number of cycles.
- Rerunning the reaction after increasing or decreasing the concentration of primers.
- Rerunning the reaction after increasing or decreasing the concentration of the template DNA.
- Checking the primers for self-complementarity and cross-complementarity.
- Checking for additional complementary regions for primers within the template DNA.
- Rerunning the reaction after increasing or decreasing the length of the primers.
- Rerunning the reaction after increasing or decreasing the annealing temperature.
- Rerunning the reaction after recalculating the primer Tm values.
C1. Golden Gate Assembly
Our parts will be assembled onto our backbone using the single-pot digestion-ligation reaction described here.
Protocol (in brief):
- Adding inserts and intact backbone into a single reaction mixture, and adding BsaI-HFv2, T4 DNA Ligase and the appropriate ATP-supplemented buffer.
- Running the reaction at 37OC for 1 hr.
- Incubating at 60OC for 5 min.
C2. Transformation and Screening
The ligated product will first be transformed into DH5α using the protocol described here.
Insert release will be validated following plasmid extraction and a double digest with BamHI-HF and XhoI in the CutSmart buffer at 37OC for 3 hours. The bands derived thusly will be resolved and visualised on a 1% agarose gel.
Protocol (In Brief):
- Subculturing overnight culture to 0.4 OD.
- Conducting a CaCl2 wash.
- Transforming the competent cells via heat shock.
- Suspending transformed cells in SOC, incubating for 3 hrs.
- Spread plating the 1:10, 1:1000 and 1:100000 dilutions of the cells on LB-Cm plates
The inserts will then be sequenced using the protocols described here.
Brief protocol for preparing the reaction mixture
- Adding the template DNA to the reaction master mix containing four nucleotides (dATP, dTTP, dCTP, dGTP) and the labelled ddNTPs (ddATP, ddTTP, ddCTP, ddGTP).
- Adding the primer and DNA polymerase.
The screening procedures described above will allow us to verify the integrity of our inserts, streamlining our future troubleshooting work.
Troubleshooting:
Preparing fresh stock of chloramphenicol. Protocol
Protocol (in brief):
Stock concentration: 25mg/mL in 100% EtOH
- Adding 2.72g chloramphenicol to a Pyrex bottle containing 80mL 100% EtOH.
- Adding the magnetic stir bar into the solution and placing the Pyrex bottle on the magnetic stirrer set at 300-600 rpm.
- Aliquoting (1mL) into microcentrifuge tubes.
C3. Transformation into Test Hosts
The plasmid will be extracted and transformed into competent E. coli K-12, SIEC and SIEC-eLEE5 using the protocol describedhere.
Protocol (In Brief):
- Subculturing overnight culture to 0.4 OD.
- Conducting a CaCl2 wash.
- Transforming the competent cells via heat shock.
- Suspending transformed cells in SOC, incubating for 3 hrs.
- Spread plating the 1:10, 1:1000 and 1:100000 dilutions of the cells on LB-Cm plates.
Troubleshooting: Detailed protocol
- Analyzing fractions saved from each step in the procedure on an agarose gel to find the source of error.
Errors
- Presence of RNA in the eluate : Adding RNase A to the resuspension buffer.
- Presence of genomic DNA in the eluate: Handling the lysate gently after addition of lysis buffer to prevent shearing and Reducing the time for lysis reaction (Maximum 5 mins).
- Little or no DNA in eluate: Checking the pH of the elution buffer (desired pH 8.5).
C4. Expression and Validation
The expression of the desired factors will be induced with L-arabinose and observed using the protocol described here. This will be performed in K-12, SIEC and SIEC-eLEE5 in the presence or absence of IPTG (to induce the expression of the T3SS).
Protocol (In Brief):
- Preparation of early-log phase secondary culture.
- Aliquoting, preserving uninduced control.
- Pelleting sample, resuspending in L-arabinose-supplemented media, incubation.
- Pelleting induced sample post-incubation, preparation, SDS-PAGE.
Performing this experiment allows us to check for the expression of our devices, making it easier to troubleshoot certain experiments in Modules 3, 4 and 5.
Troubleshooting:
- Checking the survival of K-12 in IPTG-supplemented media.
- Checking the growth curves of SIEC and SIEC-eLEE5, and E. coli K-12 in IPTG-supplemented media.
Protocol: https://pubs.acs.org/doi/full/10.1021/jacs.8b13042
5D. Transformation into Test Hosts
The ratio of GFP/mCherry fluorescence reports cleavage within FlipGFP by TEVp, while controlling for changes to global protein levels or folding states.
Testing Protocol (in brief):
- Induce expression with L-arabinose (See Module 1).
- Infect HIEC-6 with induced chassis (See Module 3).
- Measure mCherry fluorescence using spectrometric quantification (Excitation at 587 nm, Emission at 610 nm).
- Measure GFP fluorescence using spectrometric quantification (Excitation at 395 nm, Emission at 509 nm).
This allows us to directly report TEVp's differential proteolytic activity when presented with different cleavage sites, thereby informing our choice of site.
Troubleshooting:
- We will also report cleavage through Western blots and size estimation.
- We will ensure that translocation takes place using the protocols described above.
Module 6: Interface Validation
To build on the experiments we've planned, we intend to characterise our system further, and prepare it for animal trials. To do so, we will follow the workflow described here:
We have designed our previous experiments under the assumption that our system makes a certain set of functional interfaces with its target cell. We intend to validate those dependences through knocking down the host factors responsible for those interactions through transfection with shRNA after we carry out the infection protocol described in Module 3. We will observe the outcomes of these knockdowns through fractionation and Western blotting, following the protocols introduced in Module 3C.
We will use scrambled shRNA controls, to isolate responses to our specific knockdowns.
Lgr5
The CBCC marker Lgr5 is the target of our selective adhesion system. We expect that knocking it down will compromise our chassis' ability to bind to our cells. We will test this with the protocol introduced in Module 3B post transfection.
Frizzled 7
Frizzled 7 is responsible for transducing Wnt signals to the cytosol of intestinal CBCCs. Knocking it down would deprotect beta-catenin, thereby destabilising our protease and inhibiting proteolysis. We will demonstrate this effect with the protocol introduced in Module 3C post transfection.
CRM1
CRM1 is an exportin that mediates the nuclear export of proteins with the PKIA NES. Our beta-catenin-TEVp fusion has a C-terminal PKIA NES in order to retain it in the cytosol. After CRM1 knockdown, we expect proteolysis to be inhibited, due to the nuclear localisation of our protease. We will demonstrate this effect with the protocol introduced in Module 3C post transfection.
Importins Alpha and Beta
Our transcription factors will be tagged with C-terminal SV40 large T antigen NLSs, which mediate nuclear import in an importin Alpha and Beta dependent manner. Knocking them down would impair nuclear entry, which we will observe using the protocol introduced in Module 3C post transfection.
Module 7: Chassis Preparation
Chromosomal Integration
Once our parts have been rigorously tested and improved, we will insert them into defined chromosomal loci on E. coli Nissle 1917 (our desired host strain). We will carry out this procedure, involving knockins and selective marker excisions, using the protocols described here.
In parallel, we will transfer all five T3SS-associated operons from SIEC-eLEE5 to Nissle, following the protocols described here. In doing so, we will replace their IPTG-inducible promoters with a well-characterised σ70 promoter from the iGEM repository.
We will characterise this strain using the same protocols we have proposed for our engineering success workflow, using as controls empty integrants of each of our BioBricks.
Fimbriae Disruption
The specificity of our system relies, in part, on the ability of our chassis to selectively bind to Lgr5+ CBCCs. We reason that this selectivity would be enhanced by the depletion of other adhesins. We have identified Nissle's F1C fimbriae, which we plan on disrupting using antisense RNA directed at the focA gene. We will demonstrate that such disruptants would display a stronger dependence on our selective adhesion device to bind to epithelial cells, using the protocol introduced in Module 3B.
Module 8: Organoid Study
Following this characterisation, we will study the behaviour of our system in an Lgr5+ stem cell-derived human intestinal organoid. Our maintenance and infection protocols will mirror the ones followed here, and we would test the system’s response to our factors using the protocols described here.