Team:Lambert GA/Experiments

WET LAB SUMMARY

Lambert iGEM used the toehold switch mechanism to create a diagnostic method for parasitic helminths. Since we are using C. elegans as a model organism, we developed C. elegans specific toehold switch. After obtaining the toehold and trigger sequences from NUPACK and ordering them from IDT, we assembled the parts into bacterial plasmids using the Restriction Cloning workflow. The toehold switch was inserted into pSB3C5, which is a medium copy plasmid, and the trigger was inserted into pSB1A3, which is a high copy plasmid. After both the toehold and trigger are assembled into their plasmids separately, we performed a dual plasmid transformation in order for the reporter gene, GFP, to be expressed. This allows for a green fluorescence to be produced by the construct.

Building upon the 2018 Lambert iGEM part BBa_K2550000, the team also looked to improve upon the overexpression of LacZ when not induced by the corresponding trigger sequence, BBa_K2550001. The team began by modeling and testing the effect of changing Promoter sequences and Ribosomal Binding Sites on a similar construct using Biobuilder’s BBa_J10050 BBa_J10058 Promoter and RBS combinations. Those results led us to change the promoter in BBa_K2550000 from BBa_J23100 to BBa_J23106. The improved part is BBa_K2974500 and is currently, as of wiki freeze, in the cloning stage, but the team expects to update the registry with the final results after the 2019 Giant Jamboree.

WORKFLOW

The 2019 Lambert iGEM team conducted the following procedures to assemble all of the new parts; BBa_J23106 C. elegans GFP Toehold Switch, BBa_K2974400 C. elegans trigger sequence lin-4, and two parts to act as a proof of concept for the improvement of BBa_K2550000, BBa_J23106 GFP Toehold Switch and BBa_J23100 GFP Toehold Switch. After designing the new toehold C. elegans switch on NUPACK software, we ordered the parts and primers from Integrated DNA Technologies. We proceeded to perform PCRs on each of the parts. All switches and triggers were amplified and validated through gel electrophoresis. With purified PCR products of the switches, triggers, and their corresponding vectors, we digested and ligated each of these parts into their vectors through Restriction Digest Assembly. In order to clone the toehold and the trigger into their respective vectors, transformations were conducted using DH5α E. coli competent cells. We have confirmed that the right part was correctly assembled using Eurofins Genomics’s sequencing services. Dual plasmid transformations were then conducted with BL21 T7 E. coli competent cells.

C. elegans and Improved T7 Toeholds

  1. PCR
  2. Gel Electrophoresis
  3. PCR Clean-Up
  4. Digest
  5. Gel Electrophoresis
  6. Ligation
  7. Transformation/Electroporation
  8. Colony PCR and Screening
  9. Gel Electrophoresis
  10. Inoculation to Liquid Culture
  11. Miniprep
  12. Nanodrop
  13. Sequencing
  14. Dual Plasmid Transformation
  15. Inoculation to Liquid Culture
  16. Miniprep
  17. Digest
  18. Gel Electrophoresis

PROTOCOL

PCR Fragment Protocol for a 25 μL Reaction

Assemble listed reaction agents on ice

    • Q5 reaction buffer- 5 μL
    • dNTPs- 1 μL
    • Primer mix- 2.5 μL
      • 1.25 μL per primer
    • Q5 High Fidelity Polymerase- 0.25 μL
    • Template DNA- varies 1000ng
      • Lambert iGEM- 1.5 μL
    • H2O- fill to 25 μL

  1. Pipette all reaction agents into PCR tube
  2. Put PCR tube in thermal cycler and set the annealing temperature to 55℃
    • Annealing temperatures vary depending on DNA
  3. Set elongation time to 30 seconds
    • 1 min/2 kb for Q5 High Fidelity, but it may vary

PCR Vector Protocol for a 25 μL Reaction

Assemble listed reaction agents on ice

    • Q5 reaction buffer- 5 μL
    • dNTPs- 1 μL
    • Primer mix- 2.5 μL
      • 1.25 μL per primer
    • Q5 High Fidelity Polymerase- 0.25 μL
    • Template DNA- varies 1000ng
      • Lambert iGEM- 1 μL
    • H2O- fill to 25 μL

  1. Pipette all reaction agents into PCR tube
  2. Put PCR tube in thermal cycler and set the annealing temperature to 67℃
  3. Set elongation time to 1 minute 30 seconds

PCR Toehold Troubleshooting

Lambert iGEM used two methods of cloning in this year’s workflow. The first method employed Gibson Assembly, wherein Gibson primers were designed and used in PCR to create fragments of specific DNA strains and Gibson Assembly protocol was used to ligated these fragments together for subsequent transformation. The other method consisted of using high fidelity restriction digest to cut at EcoRI and PstI sites using EcoRI-HF and PstI-HF restriction enzymes. Both methods originally required PCR of target DNA, but issues with this process prevented significant success in both methods.

When using PCR on the toehold, the team expected to see a distinct band around 900-1000 base pairs when running a gel on the PCR product. Instead, there were bands at around 400 base pairs and 100 base pairs instead. The team hypothesized that some of the observed bands were the result of Primer Dimer, a by-product in PCR that occurs when primer molecules that attach to each other due to strings of complementary bases in the primers. As a result, the DNA polymerase amplifies the Primer Dimer, leading to competition for PCR reagents, thus potentially inhibiting amplification of the DNA sequence targeted for PCR amplification.

Later, the team discovered issues with hydrating primers to correct concentrations. When primer hydration was recalculated, there was observed successful PCR of the DNA products.

This gel has a 2-log ladder in lane 3 and PCR toehold in lane 4. The two bands around 400 and 100 base pairs indicate that the PCR was unsuccessful and foreign DNA was amplified.

PCR Cleanup

  1. Add 5 volumes of buffer PB to 1 volume of PCR sample and mix.
  2. If pH indicator has been added to Buffer PB check that the color of the mixture is yellow (If the color of the mixture is orange or violet, add 10 µL of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn to yellow.)
  3. Place a QIAquick spin column in a 2 mL collection tube.
  4. To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 seconds.
  5. Discard flow-through. Place the QIAquick column back into the same tube.
  6. To wash, add 0.75 mL Buffer PE to the QIAquick column and centrifuge for 30–60 seconds.
  7. Discard flow-through and place the QIAquick column back in the same tube. Centrifuge the column for an additional 1 min.
  8. Place QIAquick column in a clean 1.5 mL microcentrifuge tube.
  9. To elute the DNA, add 50 μL Buffer EB or water to the center of the QIAquick membrane and centrifuge the column for 1 min. For more concentrated DNA, add 30 μL elution buffer to the center of the QIAquick membrane. Let column stand for 1 minute then centrifuge.
  10. If purified DNA will be viewed on a gel, then add 1 volume of loading dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading gel.

Gel Electrophoresis

  1. Make 1 L of TAE buffer(1x).
  2. Add 49.5 mL of TAE buffer and .5g of agarose into a flask.
  3. Gently mix within the flask.
  4. Microwave flask for 1 minute 30 seconds on 50% power.
  5. Wait for solution to cool to 50℃.
  6. Add 8 μL of SYBR Safe into flask.
  7. Pour solution into casting tray and wait for 20 minutes or until liquid solidifies into gel.
  8. Put samples and ladder into wells.
  9. Run gel electrophoresis at 90 volts and 200 amps for 45 minutes.
  10. View results.

Gel Electrophoresis Troubleshooting

During the cloning workflow, the team faced multiple issues with gel electrophoresis. Separated DNA bands would often smear, and there would even be unforeseen fluorescence and sybr safe in different areas in the gel particularly concentrated in the wells. Additionally, some gels appeared to have DNA loaded in wells that were unloaded.

The gels did not create distinct and clear bands, and some bands or DNA was not present at all.

The wells of the gel that did not contain DNA produced fluorescence under UV light. Additionally, bands from an unknown source of DNA were found in nearly every lane.

In order to address fluorescence or SybrSafe contamination shown in unloaded wells, the team sterilized the gel combs using 90% isopropyl alcohol before gel electrophoresis. Additionally, a new stock of TAE buffer was made. Following this, wells without DNA no longer produced fluorescence under UV light. Additionally, the team decided to make a gel with a higher agarose concentration than the original 1% gel to compare their efficiency.

When the team ran both 1% and 3% gels on experimental, the results suggested that a 3% gel may actually be less reliable than a 1% gel.

This image shows the restriction digests of minipreps from two colonies labeled 1 and 3 from a transformation of pSB1A3 with RFP on a 1% gel. Well 7 has colony 1, well 8 has the 2-log ladder, and well 9 has colony 3. pSB1A3 is around 2100 bp, and RFP is around 1000 bp; these bands are clearly present in the correct locations on the 1% gel.

This image shows Restriction Digests of minipreps from two colonies labeled 1 and 3 from a transformation of pSB1A3 with RFP run on a 3% gel. Well 2 has colony 1, well 3 has the 2-log ladder, and well 4 has colony 3. The 2-log ladder is far less separated and clear, and only extremely faint bands are seen in incorrect locations for the loaded DNA.

Exonuclease

    • Transform an endA- strain with the BAC plasmid DNA and plate outgrowth onto a media plate with assigned antibiotic.
    • Incubate overnight at 30°C. Chloramphenicol levels should be maintained between 10-15 μg/ml on the selective plate.
    • Pick a colony and inoculate 10ml LB with antibiotic and incubate overnight at 30 degrees celsius.
    • Check the optical density at 600nm. It will usually be around 4 OD/ ml of cells.
    • Gather 3ml of overnight culture and purify the plasmid DNA using the Monarch Plasmid Miniprep kit.
    • To the eluted DNA add 4ul of NEBuffer 4 (10X), 4ul of 10mM ATP, and 2ul of Exonuclease V. Mix reaction and incubate at 37 degrees Celsius for 1 hour.
    • Inactivate the Exonuclease V by incubating at 70 degrees celsius for 30 minutes.
    • The plasmid DNA is ready for restriction enzyme digestion, PCR or transformation.

Restriction Digest

    • 400 ng DNA
    • 2 μl 10X CutSmart Buffer
    • 1 μl EcoRI-HF
    • 1 μl PstI-HF
    • Fill to 20 μl with Nuclease-free Water

  1. Incubate at 37℃ for 15 minutes.
  2. Heat Inactivate at 65℃ for 10 minutes.
  3. After vector digestion, Add 1/10th volume of 10X Antarctic Phosphatase Reaction Buffer to 1-5 μg of DNA digested in any restriction enzyme buffer.
  4. Add 1 μl of Antarctic Phosphatase (5 units) and mix.
  5. Incubate for 15 minutes at 37℃ for 5’ extensions or blunt ends, 60 minutes for 3’ extensions.
  6. Heat inactivate for 5 minutes at 70℃.
  7. Proceed with ligation.

Ligation

  1. Make calculations using a 3:1 molar ratio of insert to backbone.
  2. Put in each component in a microcentrifuge tube while on ice. They should be pipetted into the tube in this order: water, DNA, ligase buffer, ligase.
  3. The ligase buffer should be thawed and resuspended at room temperature.
  4. Gently mix by pipetting up and down briefly.
  5. Incubate at room temperature for 10 minutes.
  6. Heat inactivate at 65℃ for 10 minutes.
  7. Proceed to transformation.

Cell Transformation

    • Ice
    • Ice shaver
    • Styrofoam container
    • Number of microcentrifuge tubes = number of transformations + 1 extra (positive control)
    • DNA (control and experimental)
    • Waste beaker
    • Competent cells strain
    • LB
    • Specific antibiotic plates

  1. Have all materials and solutions ready
  2. Warm plates in incubator
  3. Pipette 1ul of your DNA into a labeled microcentrifuge tube
    • Use 1 μL of puc19 as control
  4. Get competent cell tube from -80F
  5. Add 50 μL of competent cell mixture to 1 μL of DNA
  6. Incubate on ice for 30 minutes
  7. Add 200 μL of warm LB/SOC media
  8. Incubate in shaking incubator for >1 hour
  9. Plate onto appropriate antibiotic plates

Inoculation into Liquid Culture

  1. Prepare Liquid LB
  2. When ready to grow culture, add Liquid LB to a tube or flask and add antibiotic to the correct concentration
    1. Create a 1:1 mL:μL ratio
    2. 5 mL LB
    3. 5 μL antibiotic
  3. Using a sterile pipette tip select a single colony from your LB agar plate
  4. Drop the tip into liquid LB and antibiotic, then swirl
  5. Cover culture with sterile aluminum foil or cap that is not airtight/li>
  6. Incubate culture at 37℃ for 12-18 hours in a shaking incubator
  7. After incubation, check for growth
  8. For long term storage continue with creating a glycerol stock

Miniprep (Omega Potocol)

    • Solution 1 (contains RNase)
    • Vortex Mixer
    • 1.5 mL microcentrifuge tubes
    • Solution 2
    • Solution 3
    • Centrifuge
    • Mini Columns
    • 2 mL Collection Tube
    • Tube Rack
    • HBC Wash Buffer diluted in isopropanol
    • Elution Buffer

  1. Grow 1-5 mL culture overnight in a 10mL-20mL culture tube.
  2. Centrifuge at 2500 xg for 10 minutes at room temperature. Decant or aspirate and discard culture media.
  3. Add 250 μL of solution 1 mixed with RNase. Vortex to mix, then transfer suspension into a new 1.5 mL microcentrifuge tube.
  4. Add 250 μL of solution 2. Invert until there is clear lysate.
  5. Add 350 μL to solution 3, insert until white precipitate forms. Centrifuge at 13,000 xg for 10 minutes.
  6. Insert mini column into 2 mL collection tube.
  7. Transfer the clear supernatant into mini column using a micropipette. Centrifuge at 13,000xg for 60 seconds. Discard filtrate and reuse collection tube.
  8. Add 500 μL of HBC Wash Buffer diluted in ethanol. Centrifuge at 13,000xg for 60 seconds. Discard filtrate and reuse the collection tube.
  9. Add 700 μL of the DNA Wash Buffer diluted in ethanol. Centrifuge at 13,000xg for 30 seconds, Discard filtrate and reuse collection tube.
  10. Centrifuge empty mini column at 13,0000xg for 2 minutes to remove ethanol.
  11. Transfer mini column to a nuclease free 1.5 mL microcentrifuge tube.
  12. Add 50 μL of Elution Buffer. Let it sit at room temperature for 5 minutes. Centrifuge at 13,000xg for 60 seconds.
  13. Store eluted DNA at -20℃.

REFERENCES

[1] Biolabs, N. E. (n.d.). Monarch® Plasmid DNA Miniprep Kit Protocol (NEB #T1010). Retrieved August 8, 2019, from https://www.neb.com/protocols/2015/11/20/monarch-plasmid-dna-miniprep-kit-protocol-t1010

[2] Biolabs, N. E. (n.d.). PCR Protocol for Taq DNA Polymerase with Standard Taq Buffer (M0273). Retrieved August 8, 2019, from https://www.neb.com/protocols/0001/01/01/taq-dna-polymerase-with-standard-taq-buffer-m0273

[3] Gootenburg, J. S. (2017). Supplementary Materials for Nucleic acid detection with CRISPR-Cas13a/C2c2. Science,4-5. Retrieved August 5, 2019, from https://science.sciencemag.org/content/sci/suppl/2017/04/12/science.aam9321.DC1/aam9321_Gootenberg_SM.pdf

[4] Hsieh, P., & New England Biolabs. (n.d.). Using Exonuclease V (RecBCD) to Eliminate Residual Genomic DNA When Purifying Low Copy Plasmids with the Monarch® Plasmid Miniprep Kit. Retrieved August 8, 2019, from https://www.neb.com/-/media/catalog/application-notes/usingexonucleasevrecbcdeliminateresidualgdnapurifyinglowcopyplasmidsmonarchplasmidminiprepkit1017.pdf?la=en&rev=4bed6f2a03f04bc4b9bd9496a8fb6ab5

[5] Inoculating a Liquid Bacterial Culture. (n.d.). Retrieved August 8, 2019, from https://www.addgene.org/protocols/inoculate-bacterial-culture/

[6] QIAquick PCR Purification Kit Protocol. (2012). Retrieved August 8, 2019, from https://static.igem.org/mediawiki/2012/a/a3/QIAquick_PCR-purification.pdf

[7] Standard RNA Synthesis (E2050). (n.d.). Retrieved August 5, 2019, from https://www.neb.com/protocols/2013/04/02/standard-rna-synthesis-e2050