Team:Lambert GA/Demonstrate

OVERVIEW

From effectively extracting genetic material from C. elegans eggs with our custom-made frugal bead homogenizer OpenCell, to using our constructed trigger to activate fluorescence in our C. elegans toehold detection system as proof of concept, LABYRINTH is widely applicable in real-world conditions. We hope to make LABYRINTH accessible to diagnostic labs all around the world.

OPENCELL (BEAD HOMOGENIZER)

C. elegans DNA Extraction

To validate the functionality and modularity of our frugal bead homogenizer, OpenCell, Lambert iGEM tested the device on plates of C. elegans eggs. Inserting the eggs into the tubes and homogenizing it at above 1200 RPM, we analyzed the resulting concentration and purity of the extracted genetic material.

For further confirmation, Lambert iGEM sent OpenCell to the Georgia State University (GSU) iGEM team. The device was tested on microalgae and spinach leaf tissue, and the DNA yield and efficiency were compared to that of a commercial homogenizer. OpenCell’s accompanying frugal beads were also tested against commercial Qiagen-modeled beads. Variables such as time and number of beads were adjusted to analyze the resulting concentration and purity yield for optimal results.

The GSU iGEM team provided significant feedback on ease of use, functionality, and potential improvements to our design. After testing, they described our frugal homogenizer results as “better than the commercial homogenizer”, as it resulted in greater cell viability. We hypothesized that our homogenizer was less aggressive than the commercial one with more tunability, making OpenCell suited for a larger range of organisms.

Homogenization Method Yield (μg) Purity (A260/A280)
Vortex Mixer-Quiagen Garnets 0.135 1.37
OpenCell-Zirconium Beads 100 Micron 0.276 1.62
OpenCell-Quiagen Garnets 0.69 1.77
OpenCell-Quiagen Garnets after PCR Purification 1.560 1.74

The DNA extraction from C. elegans eggs was significantly better after the adjustments to the protocol as well as extending the time of homogenization. As shown above in the results, Opencell worked much better in breaking through the chitin layer in comparison to the regular vortexer.


DNA to RNA

Gel with amplified DNA from PCR in lane 4 and a 100bp ladder in lane 8. The band around the 100 base pair band on the ladder indicates a successful PCR


After extraction of DNA from the C. elegans, it was amplified by PCR. The entire genomic DNA of the C. elegans was extracted in the sample, but the PCR was only done with the forward and reverse primers of the trigger sequence to the toehold. A gel was run to confirm the PCR. The gel proved that the amplification of the trigger sequence was successful. After the PCR, a PCR purification was done using the Monarch® PCR & DNA Cleanup Kit (5 μg) in order to purify the amplified DNA.

In order for translation of the reporter protein to occur, a complementary sequence of RNA must first bind to the hairpin stem and unravel the loop. Only then will the ribosomal binding site and start codon be exposed, allowing translation. In our process, the 3D-printed filter and bead homogenizer allowed us to isolate the helminth DNA, but this product must then be converted into RNA for the reporter to be expressed. The isolated DNA will be appended with a T7 promoter sequence and annealed to a short T7 primer. Then, the construct will be incubated with T7 polymerase overnight at 37°C using the HiScribe T7 Quick High Yield RNA Synthesis Kit. The resulting RNA will then be purified using RNAXP clean beads at 2x ratio of beads to reaction volume, with an additional 1.8x supplementation of isopropanol.

C. ELEGANS TOEHOLD PROOF OF CONCEPT

Construction

This year, Lambert iGEM constructed a C. elegans toehold construct as proof of concept to circumvent working with infectious materials from target parasitic helminths. We used a Green Fluorescent Protein (GFP) reporter system to indicate the presence of helminth eggs in fecal samples through the extracted complementary RNA trigger.

The toehold switch is an RNA riboregulator that consists of a switch RNA hairpin and a complementary trigger RNA. In the event that the trigger binds to the switch, the system unravels and allows for translation of the GFP gene. We measure and quantify the fluorescence using FluoroCents.

The construct above displays the proof of concept BBa_J23106 GFP switch and T7 trigger. The switch consists of a medium constitutive promoter with GFP as the reporter gene.

The toehold part (BBa_K2974316) is assembled in pSB3C5, which is a medium-copy plasmid. The T7 C. elegans trigger (BBa_K2974400) is assembled in pSB1A3, which is a high-copy plasmid. After constructing these parts separately, we performed a dual plasmid transformation in order to obtain the trigger in the presence of the toehold.

Results

Toehold Switch Assembly

The C. elegans Toehold GFP BioBrick (BBa_K2974316) was assembled into pSB3C5, a medium-copy plasmid, using the Restriction Cloning Workflow with protocol provided by New England Biolabs (NEB).

After obtaining the C. elegans Toehold GFP gBlock, we used Polymerase Chain Reaction to amplify the amount of DNA in order to obtain a higher concentration for the rest of our workflow. We then digested the Toehold insert using the restriction enzymes EcoRI-HF and PstI-HF. We confirmed that the insert digest worked using gel electrophoresis.

In the confirmation gel, there is a faint band signifying DNA around 500 bp. This band represents the Toehold Switch insert. There is a 2-log ladder in lane 3.



We ligated the Toehold insert digest with a pSB3C5 vector digest, previously digested with the Leaky Toehold workflow, and proceeded to transform using DH5α E. coli competent cells on chloramphenicol-resistant LB plates. After growing in an incubator at 37ºC for 24 hours, we analyzed colony growth.


This plate shows the transformed C. elegans Toehold Switch with pSB3C5 in DH5α E. coli competent cells.




We then proceeded with DNA purification using the Omega Miniprep Kit. After obtaining Toehold DNA, we sent the purified DNA to Eurofins Genomics for sequencing. Results came back successful.



In lane 7, we ran a 2-log ladder. In lane 8, there are faint bands around 3000 bp long and 500 bp long. This shows that the Toehold insert was successfully assembled into pSB3C5.



Additionally, we confirmed that the Toehold insert was successfully assembled into pSB3C5 by performing a Restriction Digest with the Toehold Miniprep product. We ran a confirmation gel, and we obtained successful results.


We aligned our Eurofins Genomics sequencing results with our ordered Toehold Switch insert. The results were successful.



After assembling the Toehold insert into pSB3C5, we moved on to assembling the C. elegans trigger into pSB1A3.

Trigger Assembly

The T7 C. elegans Trigger (BBa_K2974400) was assembled into the high copy plasmid, pSB1A3, using the Restriction Cloning Workflow with protocol provided by New England Biolabs (NEB).

After rehydrating the C. elegans Trigger gBlock, we used Polymerase Chain Reaction to amplify the amount of DNA in order to obtain a higher concentration for the rest of our workflow. The trigger was then digested using EcoRI-HF and PstI-HF, and we ran a gel showing a band around 100-200 base pairs, confirming the presence of the insert.

The gel electrophoresis demonstrates the successful Trigger insert digest. In lane 4 and 6, the 2-log ladder and the 100bp ladder are present, respectively. The trigger insert band, in lane 5, is faintly shown around 160 bp.




With the presence of the insert confirmed, we proceeded with a ligation between the trigger and a psB1A3 vector digest. This was later transformed using DH5α E. coli competent cells, and after 24 hours of colony growth at 37ºC, an analysis of the carbenicillin plates showed that the ligation was successful.



The white trigger colonies can be seen on this plate, showing that the trigger successfully ligated into pSB1A3. There is some RFP contamination present.





From there, we chose three colonies to inoculate into liquid culture, and then we processed with DNA purification using the Omega Miniprep Kit. After obtaining isolated Trigger DNA, it was sent to Eurofins Genomics for sequencing. Sequencing results came back successful!



We aligned the Trigger insert with the Eurofins Genomics sequencing results. This showed that the Trigger was successfully cloned.

Dual Plasmid Transformation

Lambert iGEM used the Miniprep products from both the Toehold Switch Assembly and the Trigger Assembly to perform a dual plasmid transformation on carbenicillin/chloramphenicol-resistant LB plates. We decided to use BL21(DE3) E. coli competent cells from NEB because they allow for T7 expression. After growing for 48 hours in the incubator at 37ºC, we observed GFP expression from the transformed dual plasmid colonies.

Dual Plasmid transformation colonies are shown here. Since the toehold was now in the presence of the trigger, the competent cells expressed a green fluorescence.



We then continued to miniprep from the LB liquid cultures with carbenicillin and chloramphenicol resistance. After obtaining a purified DNA product of the Dual Plasmid, we performed a Restriction Digest in order to demonstrate that both vectors were successfully cloned into BL21 (DE3) E. coli competent cells. After viewing results using gel electrophoresis, we confirmed that the dual plasmid transformation was successful.

This is the Dual Plasmid Transformation of T7 Trigger Sequence and T7 Toehold GFP. The gel serves as confirmation from Restriction Digest. These images show the expected results of our constructs.



FluoroCents, the low-cost fluorometer developed by 2019 Lambert iGEM, was then used to confirm that the dual plasmid transformation successfully produced a green fluorescence by comparing the mean lux values between the toehold switch, trigger, and the dual plasmid. Results came back successful.

FluoroCents’ Use in Lambert iGEM’s LABYRINTH

C. elegans Toehold Switch and Trigger Verification

FluoroCents was used in this year’s iGEM project as part of the Wet Lab process, specifically to verify that our C. elegans toehold was functional. If the cells were transformed with either the trigger sequence or the toehold switch plasmid separately, they should not fluoresce. However, in a dual plasmid transformation, the cells should fluoresce, as the trigger is present and would activate our toehold switch.

Thus, FluoroCents was used in order to detect any presence of fluorescence. The results obtained from our testing are below:

This graph compares the mean lux values of the C. elegans toehold switch, trigger, and the dual plasmid. The toehold switch and trigger have very similar lux values to LB and plain cells, whereas the dual plasmid has a higher lux value, indicating fluorescence.



From the relative comparisons of the cell types shown, the culture of cells with our C. elegans toehold and trigger separately did not appear to fluoresce because their lux values fell in the range of the values of LB/Plain cell mean lux. However, the dual plasmid transformation had a mean lux value with an error range above all the other cell types, including the plain cells and each of the individual trigger and toehold cell cultures, signifying that fluorescence was present and that the C. elegans toehold switch and trigger together functioned properly.

Leaky Toehold Improvement Verification

As an improvement from last year’s project, Lambert iGEM used a different promoter this year, BBa_J23106, instead of last year's promoter, BBa_J23100, in our toehold constructs in order to prevent the leakiness that existed in last year’s project. The cells with the old promoter would fluoresce and have a mean lux above the plain cells and cells with the trigger due to leakiness, and the cells with the new promoter will not fluoresce and will have similar mean lux to the plain cells and cells with the trigger. Both dual plasmid transformations with both promoters should simply have mean lux values above their respective promoter cell types as a positive control to show the switch/trigger is functional. The data collected is shown below.

The data represents a comparison of lux values between promoters BBa_J23100 and BBa_J23106 in strains of plain LB, plain E.coli cells, trigger-pSB6A1, toehold-pSB3C5, and dual plasmid transformation.



From this data, we can see the trigger-transformed cells for both promoters have similar lux values and overlapping error ranges with the plain cells, as expected. The dual plasmid cells for both promoters have mean lux and error ranges outside of both their respective toehold only and trigger only cells, signifying that the system is functional. The toehold only cell with the old promoter did have a higher mean lux value than the trigger and plain cells, signifying fluorescence and leakiness, while the new toehold cells matched the mean lux values and error ranges of the trigger and plain cells, signifying that the new promoter construct did not fluoresce and was not leaky. However, the error bars for the old promoter toehold cells do overlap with the toehold, so one cannot say with certainty that there was leakiness in the old promoter and that the new promoter removed this leakiness without performing more trials.

BIOSENSOR CELLS

Freeze Drying Protocol

These protocols are for using the Biosensor Cells in the field

  1. These protocols are for using the Biosensor Cells in the field.
  2. Add 5 ml of sterile LB media and 5ul of chloramphenicol and ampicillin to a culture tube.
  3. Obtain the desired plate. Take 1-3 colonies from the biosensor cells’ plate with a sterilized inoculating loop. Stir vigorously to ensure colonies are dispensed into solution.
  4. Place culture tubes on a rack in the incubator. Set to shake and temperature to 36-38 C, and grow overnight. Centrifuge liquid culture and discard the supernatant. Resuspend the pellet in 5ml of Microbial Freeze Drying Buffer with tryptic soy broth.
  5. Aliquot 500ul of the suspension into sterile vials with stopper.
  6. Turn on the lyophilizer and start the condenser. Set the shelf to 4°C.
  7. Center the vials on the shelf. Either manually or with programmed controls, freeze the samples down to -40°C. This should take about 30-60 minutes, but it is very dependent upon the lyophilizer. If the rate of freezing can be controlled, a practical rate is to drop the temperature by 1°C per minute. The samples should be visually frozen.
  8. Let the samples sit at -40°C for 1 hour to complete freezing.
  9. Turn on the vacuum pump. Within 10-20 minutes, the vacuum should be under 200 millitorr (mtorr).
  10. After the vacuum is below 200 mtorr, increase the temperature of the shelf for primary drying. The temperature can be up to -15°C. Let continue overnight.
  11. For second drying, raise the shelf temperature to 20°C and dry for 2 hours.
  12. With the stoppering mechanism, pit the stoppers on the vacuum. Turn off vacuum.
  13. Store at 4°C in the dark.

In-Field Preparation of Cells

Tubes should be on ice at all times unless centrifuging

  1. Resuspend in 5 ml of LB agar. Incubate for two hour.
  2. Aliquot 1 ml of that liquid culture into 4 ml of sterile LB.
  3. Shake in an incubator overnight. If no incubator present, let sit at room temperature for a day and a half.
  4. Add the 5 ml liquid culture into 500 ml of sterile LB.
  5. Using the portable spectrophotometer, check the optical density every hour. Continue growing until the OD is 0.6.
  6. Pour cells into 50 ml conical tubes, on ice. Keep there for 30 minutes.
  7. Pipet up the bottom 1.5 ml in the tube and transfer to a microcentrifuge tube.
  8. With the 3D-fuge, centrifuge cells for 20 minutes, discard supernatant.
  9. Add 1.5 ml of ice-cold sterile into the microcentrifuge to resuspend. Add into empty 50 ml conical tube.
  10. Add another 11.5 ml of ice-cold sterile milliQ water to each conical tube, pipet up and down.
  11. Combine samples into 2 total conical tubes.
  12. Let sit on ice for 30 minutes. Take bottom 1.5 ml of each sample and transfer into a empty microcentrifuge tube.
  13. With 3D-fuge, centrifuge for 20 minutes, discard supernatant, and repeat steps 9 and 10.
  14. Let sit for 30 minutes on ice.
  15. Transfer bottom 1.5 ml into a microcentrifuge tube. Centrifuge for 20 minutes, discard supernatant, and add 1.5 ml of ice-cold 10% glycerol.
  16. Transfer the microcentrifuge into an empty conical tube. Add 23.5 ml of ice-cold 10% glycerol.
  17. Let sit for 30 minutes on ice. Repeat steps 15 and 16.
  18. Let sit for 30 minutes on ice. Take bottom 1.5 ml and transfer into a microcentrifuge tube. Centrifuge with 3D-fuge for 20 minutes.
  19. Resuspend in 1.5 ml of ice-cold 10% glycerol. Transfer into conical tube and add 2.5 ml of ice-cold 10% glycerol.
  20. Aliquot 1 ml into microcentrifuge tubes. Store in -80 freezer.

References

[1] 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.

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