Team:MEPhI/Project

Project

Lab Strategies

Hypothesis

The proteins that are highly expressed in inhabitants of high background-radiation areas have radioprotective properties, and they can be used to protect humans against continuous low dose radiation. Gut microbiota is a good target to edit and reproduce these radioprotective proteins.


Objective

Creating a protective barrier against continuous low-dose radiation for humans through a genetic modification of the gut microbiota, which would lead to the production of radioprotective proteins.


Overview

The aim of our iGEM project was to demonstrate the operability of our hypothesis, according to which bacterial cells typical for the human gut can produce proteins that perform a radioprotective function for human cells.


February
We analyzed proteins that have been reported as highly overexpressed in inhabitants of high background-radiation areas and chose two of the most expressed ones: Wnt-10a and Bloom syndrome protein. Using protein blast software we also evaluated which human proteins are homologous with respect to proteins that perform a radioprotective function in other organisms, such as tardigrades (Dentin sialophosphoprotein, Mucin-21, Hornerin).

The proteins BLM and Wnt-10a were selected to develop the technology of testing proteins for radioprotective properties. Even though Dentin sialophosphoprotein, Mucin-21, Hornerin were considered as potential targets in our project, we refused to test them at the first stage of the project in order to pay more attention to the delivery of genetic constructs and the safety of their use.


March
Our design vector was based on pET23b plasmid, which includes the ampicillin resistance gene. We plan to eliminate antibiotic resistance genes in our plasmid constructs in order to avoid the uncontrolled spread of these genes in the intestinal microbiome and in the environment. However, antibiotic resistance genes are needed at the design testing stage. We replaced the ampicillin resistance gene in the initial plasmid with a kanamycin resistance gene (which is easier to work with) but what is more significant, we needed to make sure that we could do the cloning in those laboratories that are available to our team.
Therefore, we were pleased to see on the sequence that the kanamycin resistance gene was successfully inserted into the vector.


April
Solving the problem of guaranteed plasmid inheritance.
Since we plan to use our genetic vectors as part of the gut microbiota, it was important for us to solve the problem of inheritance of the plasmid vector. After analyzing the literature sources, we selected three types of inserts for testing that guarantee plasmid inheritance.


We have cloned into our vector three types of inserts, each of which should provide inheritance of the plasmid during bacterial cell division:


  1. partition locus from the pSC101 plasmid
  2. partition locus Park
  3. Axe-The toxin-antitoxin system

For safety reasons, we have abandoned the plasmid based on the toxin-antitoxin system. Also, according to our data, the cell culture grew at a higher rate in the presence of the partition locus from the pSC101 plasmid. That is why we chose this particular locus for use in all subsequent constructions. Starting from this stage, we gave to our vector its own name pMISS.


May
In May, we embarked on an expedition to the Bryansk region (Russia) to determine those populated areas that retained a high radiation background after the 1986 Chernobyl disaster. We found out that even after many years, many people live in areas with a high level of ionizing radiation and made a map of the contaminated areas. We planned to collect biological material from local residents in order to independently analyze their transcriptome, but we encountered legal obstacles.
Another part of the team was engaged in the design of primers for PCR and amplification of the selected 9 targets for insertion into the genetic construct.


June
In June, part of the group was engaged in cloning selected targets and succeeded in obtaining vectors containing two proteins potentially possessing radioprotective properties: Wnt10a and BLM.
We also conducted interviews with potential users of our system and realized that it is not secure enough in terms of control. Therefore, it was decided to add an inducible promoter to the genetic vector, which was chosen as an arabinose promoter (safe for human cells).


July
In July, it becomes clear that we would not have time to clone all the selected targets and decided to focus on testing the constructs that we have, but we tried to add an arabinose promoter to the constructs instead of the standard T7 promoter. This part was taken from the plasmid provided by Vasily N. Stepanenko, the head of Experimental Biotechnological Production IBCh RAS, Miklukho-Maclay Str., 16/10 Moscow, Russia.
In addition, we realized that the delivery of some elements of our system into human cells may not be very effective and tried to find an alternative delivery method. It turned out that silicon nanoparticles are well suited for our purposes.
In July, we tested several ways to modify the silicon surface. In the engineering success section, we described only relatively successful attempts and did not list the failed attempts in which we tried to place RNA inside porous silicon particles using ultrasound.


August
Our main experiments in August focused on studying the rate of desorption of siRNA from the surface of nanoparticles with various types of modified surfaces. Alina and Nastya managed to get good results, which showed that RNA binds well to nanoparticles. In addition, the nanoparticles were tested for biocompatibility using the MTT test on Hek293T and MSC cell lines.
We also managed to incubate nanoparticles loaded with siRNA with MSC cells. From MSC cell culture using the miRNeasy Serum / Plasma Kit total RNA was isolated and converted to cDNA using the QuantiTect Reverse Transcription Kit. We also ordered primers to understand whether our nanoparticles penetrate cells and whether microRNA is able to influence the expression of the target genes that we selected, but we faced an unexpected problem: it turned out that the primers that we ordered and paid for would not arrive until a month later, as the company is busy synthesizing the primers for testing the coronavirus.


September
In September, we had a lot of work outside the laboratory. It was necessary for us to come to an agreement with the M. Tsyba, so that they provide us with a technological opportunity to irradiate cells and issue the necessary passes for students. We were almost unable to progress with the project, except for obtaining the final form of constructs containing two target proteins ( Wnt10a and BLM) under the control of an arabinose promoter. We also reviewed the literature to determine the optimal method for incubating human cells with bacterial cells in order to test the radioprotective potential of the genetic constructs we obtained.


October
In October, we finally managed to test the obtained genetic constructs for radioprotective properties, however, apparently, we were too optimistic about the capabilities of our system and irradiated the cells with too high doses (20 Gray). As a result, we have not yet been able to find the threshold values at which our system gives an optimal result. We are continuing this work at the present time.


Protocols

Here are all the protocols we used during the work on our project. If you want more information you can click on the link to see the complete protocol. If you have any questions, feel free to contact us n.anasttasia@gmail.com


Agarose gel electrophoresis
Materials

  • Agarose
  • 1X TAE
  • Ethidium bromide (stock concentration of 10 mg/mL)

Methods

  1. Mix 0.6 g of agarose powder with 40 ml 1X TAE in a microwavable flask to get a final concentration of 1.5% (w/v).

    Note: Do not mix with water and make sure to use the same buffer as the one in the gel box.
  2. Microwave pulses of 30-45 sec until the agarose is completely dissolved.

    Note: Do not over boil, it will change the agarose concentration.
  3. Let agarose solution cool down to about 50°C, usually when you can keep your hand on the flask.
  4. Add ethidium bromide (EtBr) to a final concentration of approximately 0.2-0.5 μg/ml. EtBr binds to the DNA and allows you to visualize the DNA under ultraviolet (UV) light.

    Pro-Tip: Usually about 2-3 μl of lab stock solution per 100 ml gel.

    Caution: EtBr is mutagen, wear gloves, eye protection and lab coat.
  1. Pour slowly the agarose into a gel tray with the well comb in place to avoid bubbles. If bubbles are formed, can be pushed away with a pipette tip.
  2. Place newly poured gel at 4°C for 10-15 mins OR let sit at room temperature for 20-30 minutes, until it has completely solidified.

Loading Samples and Running an Agarose Gel

  1. Add loading buffer to each of your DNA samples.

    Note: Loading buffer serves two purposes: 1) it provides a visible dye that helps with gel loading and allows you to gauge how far the DNA has migrated; 2) it contains a high percentage of glycerol that increases the density of your DNA sample causing it settle to the bottom of the gel well, instead of diffusing in the buffer.
  2. Once solidified, place the agarose gel into the gel box (electrophoresis unit).
  3. Fill gel box with 1X TAE (or TBE) until the gel is covered.

    Pro-Tip: Remember, if you added EtBr to your gel, add some to the buffer as well. EtBr is positively charged and will run the opposite direction from the DNA. So if you run the gel without EtBr in the buffer you will reach a point where the DNA will be in the bottom portion of the gel, but all of the EtBr will be in the top portion and your bands will be differentially intense. If this happens, you can just soak the gel in EtBr solution and rinse with water to even out the staining after the gel has been run, just as you would if you had not added EtBr to the gel in the first place.
  4. Carefully load a molecular weight ladder into the first lane of the gel.
  5. Carefully load your samples into the additional wells of the gel.
  6. Run the gel at 90V until the dieline is approximately 75-80% of the way down the gel during 1 hour.

    Note: Black is negative, red is positive. The DNA is negatively charged and will run towards the positive electrode. Always Run to Red.
  7. Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box.
  8. Using any device that has UV light, visualize your DNA fragments.

    Pro-Tip: If you will be purifying the DNA for later use, use long-wavelength UV and expose for as little time as possible to minimize damage to the DNA.

PureLink® HiPure Plasmid Filter DNA Purification Kit

Before starting

  • Add RNase A to Resuspension Buffer (R3) according to the instructions on the label.
  • Warm Lysis Buffer (L7) briefly at 37°C to redissolve any particulate matter, if necessary.

Midiprep Procedure
Notes

  • For all column steps, use Column Holders (included) or a Nucleic Acid Purification Rack.
  • Grow transformed E. coli in LB medium. Use 15–25 mL (high copy number plasmid) or 25–100 mL (low copy number plasmid) of an overnight culture.

Isolate midiprep plasmid DNA

  1. Equilibrate. Apply 15 mL Equilibration Buffer (EQ1) directly into the Filtration Cartridge, which is inserted in the PureLink® HiPure Midi Column. Allow the solution in the column to drain by gravity flow.
  2. Harvest. Centrifuge the overnight LB culture at 4000 × g for 10 minutes in a 50-mL disposable centrifuge tube. Remove all medium.
  3. Resuspend. Add 10 mL Resuspension Buffer (R3) with RNase A to the cell pellet and gently shake the pellet until the cell suspension is homogenous.
  4. Lyse. Add 10 mL Lysis Buffer (L7). Mix gently by inverting the capped tube until the mixture is homogeneous. Do not vortex. Incubate the tube at room temperature for 5 minutes.
  5. Precipitate. Add 10 mL Precipitation Buffer (N3). Mix by inverting the tube until the mixture is homogeneous.
  6. Clarify. Transfer the precipitated lysate into the column. Allow the lysate to drain by gravity flow. Optional: Wash the column with 10 mL Wash Buffer (W8). Allow the buffer to flow through the column by gravity flow.
  7. Wash. Discard the inner filtration cartridge. Add 20 mL Wash Buffer (W8) to the column. Discard the flow-through after Wash Buffer (W8) drains from the column.
  8. Elute. Place a sterile 15-mL centrifuge tube under the column. Add 5 mL Elution Buffer (E4) to the column. Allow the solution to drain by gravity flow. Discard the column. The elution tube contains the purified DNA. Proceed to Precipitate DNA.

Precipitate DNA

  1. Precipitate. Add 3.5 mL isopropanol to the eluate. Incubate DNA with isopropanol for 2 minutes at room temperature. Centrifuge the tube at >12,000 × g for 30 minutes at 4°C.
  2. Wash. Discard the supernatant. Add 3 mL 70% ethanol to the pellet. Centrifuge the tube at >12,000 × g for 5 minutes at 4°C. Remove the supernatant.
  3. Resuspend. Air-dry the pellet for 10 minutes. Resuspend the purified DNA in 100–200 μL TE Buffer (TE). Store plasmid DNA at −20°C.

Preparation of chemically competent cells

Materials

  • Plate of cells to be made competent
  • TSS buffer
  • LB media
  • Ice

Preparation


  1. Inoculate 5 ml LB medium with the E. coli strain of which you want to make competent cells and incubate overnight at 37°C.
  2. Use the overnight culture to inoculate 500 ml LB 1 /100 (50 ml + 450 ml of medium)
  3. Incubate 3 hours at 33 -34°C. Prepare 7 falcons tubes with 3.5 ml medium
  4. Chill on ice the culture for at least 10 min.
  5. Set up the centrifuge at 4000 rpm
  6. Pre-cool 24 pp 1.5ml
  7. Aliquot cells in 1 ml tubes
  8. Centrifuge at 3000 rpm for 10 min at 4ªC
  9. Select a supernatant
  10. Ice-cold the TSS buffer
  11. Suspend precipitation in the TSS buffer - 10% of the initial volume kepping the cells in ice.
  12. Add 100 µl TSS
  13. Flash freeze the cell suspension in liquid nitrogen and store the tubes at -80°C.
  14. Use immediately cells for their transformation

To make 50 mL TSS buffer:
5 g PEG
0.3 g MgCl2x5H2O
2,5 ml DMSO
add LB to 50 ml


Filter sterilize (0.22 μm filter) If using non-chemically resistant filters (e.g., cellulose nitrate), add DMSO after sterilization. DMSO should be sterile in and of itself, so it may be prudent it add it afterward if you are unsure about the compatibility of your filters


Store at 4ºC.

Transformation Protocol


  1. Add 10 ml of DNA to 200 ml of competent cells and chill in ice for 20 minutes. (for cultures BL21 and JM109, in no case should antibiotics be added to the medium, when using the Rosetta 2 (DE3) culture, chloramphenicol can be used)
  2. Place the cells in a water bath or a 42ºC solid-state thermostat for 3-5 minutes
  3. Transfer the cells to the ice for 10 minutes.
  4. Add 800 ml of warm SOC medium and incubate on shaker at 37ºC for 1 hour.
  5. Add 300-500 µl transformed cell suspension onto LB agar plates with the appropriate antibiotic (in our case kanamycin, working concentration 100 ug/mL).
  6. After 16-18 hours, separate individual colonies and transfer to a liquid medium SOC.
  7. Incubate in LB medium with the appropriate antibiotic for 18-22 hours in an incubation shaker at 180 rpm, 37°C

Restriction Digest

Digest Mix
0.2-1μg DNA from prep or PCR
10% (by volume) Restriction Buffer
1% (by volume) BSA stock
2.5% - 5% (by volume) of each restriction enzyme.
ddH2O to produce correct percentages by volume
Enzymes < 10% of total volume so glycerol is < 5% of total volume.

Reaction conditions:
Incubate reaction at 37°C for at least 1 hour; longer digest gives more complete digestion, especially if you have >1µg DNA, but can at times give nonspecific digestion, even if glycerol is <5%. Heat kill enzymes at 80°C for 20 min. Store at -20°C.

DNA ligation

Reagents
  • T4 DNA ligase
  • 10x T4 DNA Ligase Buffer
  • Deionized, sterile H2O
  • Purified, linearized vector (likely in H2O or EB)
  • Purified, linearized insert (likely in H2O or EB)

Equipment
Vortex


Procedure:
10μL Ligation Mix

Larger ligation mixes are also commonly used
  • 1.0 μL 10X T4 ligase buffer
  • 6:1 molar ratio of insert to vector (~10ng vector)
  • Add (8.5 - vector and insert volume)μl ddH2O
  • 0.5 μL T4 Ligase

Calculating Insert Amount

Insert Mass in ng=6×(Insert Length in bp/Vector Length in bp)×Vector Mass in ng

The insert to vector molar ratio can have a significant effect on the outcome of a ligation and subsequent transformation step. Molar ratios can vary from a 1:1 insert to vector molar ratio to 10:1. It may be necessary to try several ratios in parallel for best results.


Method
  1. Add appropriate amount of deionized H2O to sterile 0.6 mL tube
  2. Add 1 μL ligation buffer to the tube.
    Vortex buffer before pipetting to ensure that it is well-mixed.
    Remember that the buffer contains ATP so repeated freeze, thaw cycles can degrade the ATP thereby decreasing the efficiency of ligation
  3. Add appropriate amount of insert to the tube.
  4. Add appropriate amount of vector to the tube.
  5. Add 0.5 μL ligase.
    Vortex ligase before pipetting to ensure that it is well-mixed.
    Also, the ligase, like most enzymes, is in some percentage of glycerol which tends to stick to the sides of your tip. To ensure you add only 0.5 μL, just touch your tip to the surface of the liquid when pipetting.
  6. Let the 10 μL solution sit at 22.5°C for 30 mins
  7. Denature the ligase at 65°C for 10min
  8. Store at -20°C

PCR

  1. Set all reagents on ice.
  2. Gather reaction mix into 50 µL volume in a thin walled 0.2 ml PCR tubes.
  3. Add reagents in following order:
    • Water (to 50 µL)
    • Buffer (10 x)
    • dNTPs (200 µM)
    • Template primers (200 pg/µL)
    • Taq polymerase (0.05 units/µL)
    • DMSO (optional, 1 to 10% w/v)
  4. Gently mix by tapping tube. Briefly centrifuge to settle tube contents.
  5. Prepare negative control reaction without template DNA.
  6. Prepare positive control reaction with template of known size and appropriate primers.
  7. Set up the thermocycler for PCR reaction:
    • Initial Denaturation at 94°C for 5 min.
    • Denaturation (94°C, 30 sec, ×30-35)
    • Primer Annealing (Tm-5°C, 45 sec, ×30-35)
    • Extension (72°C, 1 min per kb, ×30-35)
    • Final Extension (72°C, 5 min)
  8. Analyze the results via gel electrophoresis.

Testing the radioprotective activity of genetic constructs expressing mRNA for proteins Wnt10a and BLM

  1. Cells of RPMI 8866 Human Cell Line were used in dosage of 500 000 cells per well and precultured in 1 ml of cell culture media (PRMI 1688 medium + 10% FBS).
  2. Conditioned media of gene-engineered bacterias (WNT and BLM) were filtered (0.22 µm micropores) to eliminate living bacterias and added to the wells with RPMI 8866 cells as 1:100.
  3. cell cultures were exposed to 20 Gy Beta-irradiation.
  4. The evaluation of cell viability was performed after two days using LIVE/DEAD Assay (Ethidium homodimer-1 and Calcein AM) and confocal microscope