Team:Lambert GA/Results - 2019.igem.org

C. elegans Toehold Switch

Toehold Switch Assembly

The C. elegans Toehold GFP BioBrick (BBa_K2974316) was assembled into pSB3C5, which is 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, Polymerase Chain Reaction was used to amplify the amount of DNA in order to obtain a higher concentration for the rest of our workflow. Toehold insert was digested using restriction enzymes, EcoRI-HF and PstI-HF. The insert digest was confirmed to work using gel electrophoresis.

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

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

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

Next, DNA was purified using the Omega Miniprep Kit. After obtaining Toehold DNA, the purified DNA was sent to Eurofins Genomics for sequencing. Results came back successfully.

In lane 7, a 2-log ladder was run. 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 have gotten successful results.

Eurofins Genomics sequencing results aligned 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 respectively are present. The trigger insert band, which is 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 then sent to Eurofins Genomics for sequencing. Sequencing results came back successful!

The Trigger insert was aligned 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 is 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.

(Top) This is the Dual Plasmid Transformation of T7 Trigger Sequence and T7 Toehold GFP (Bottom) Gel for confirmation from Restriction Digest. These 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.

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.

IMPROVED PART

BBa_J23106 Promoter

Transformation of BBa_K2974300

The transformation of the team’s composite part BBa_K2974300, the BBa_J23106 (medium-strength) promoter with the Toehold eGFP construct, with pSB3C5, did not produce green fluorescent colonies. The absence of fluorescence was ideal, as the reporter protein eGFP should only be expressed in the presence of the trigger sequence. The red fluorescent colonies seen in both images is a result of the backbone, pSB3C5, which was originally cloned with Red Fluorescent Protein (RFP). Lambert iGEM obtained this stock of pSB3C5 from the iGEM Distribution Kit and proceeded to digest the plasmid with High Fidelity EcoRI and PstI restriction enzymes the red fluorescence observed on each plate indicates that the plasmid re-ligated to the RFP insert originally present in the pSB3C5 stock instead of the toehold construct during ligation with T4 ligase. The team inoculated colonies that were not producing RFP which appeared translucent after a 24 hour incubation period. Sequencing then confirmed that colony 3 included the correct toehold sequence.

(Left) This is the transformation of the part BBa_K2974300, BBa_J23106 promoter Toehold eGFP construct, not under ultraviolet (UV) light. (Right) This is the same transformation of part BBa_K2974300 under UV light.

This is the 1% gel with the 2-log ladder in well 5 and colony 3 in well 6. Both the insert (toehold - approximately 900 base pairs) and the vector (pSB3C5 - approximately 3000 base pairs) are present in colony 3. When this colony was sent out for sequencing, it aligned with the desired toehold sequence.

This is the picture of the sequencing results from IDT confirms that the toehold construct assembled with pSB3C5 aligns with the desired toehold sequence.

Dual Plasmid Transformation of BBa_K2974300 with BBa_K2550001

The dual plasmid transformation of the part BBa_K2974100, in pSB3C5, and part BBa_K2550001, the trigger sequence, in pSB6A1, produced green fluorescent colonies. The team decided to plate the transformations on two plates with different antibiotics - chloramphenicol/ampicillin and chloramphenicol/carbenicillin - as plasmid pSB6A1 allows for transformed E. coli cells to gain both ampicillin and carbenicillin antibiotic-resistance. Both of the dual plasmid transformations produced low-grade green fluorescence in colonies, which indicated a successful binding of the toehold and trigger sequence and expression of eGFP. There was a 48 hour incubation period required to produce fluorescence.

This shows the dual-plasmid transformations of the BBa_J23106 Toehold construct (plate 1 containing carbenicillin/chloramphenicol on the left and plate 2 containing ampicillin/chloramphenicol on the right) of the toehold assembled with pSB3C5 and the trigger assembled with pSB6A1. A successful binding of the toehold and the trigger shows low-grade fluorescence from the expression of eGFP.

BBa_J23100 Promoter

Transformation of BBa_K2974700

The transformation of the team’s composite part BBa_K2974700, BBa_J23100 (strong) promoter and Toehold eGFP construct, in pSB3C5, produced both translucent and red pigmented and fluorescing colonies. The red fluorescent colonies seen in the image is a result of the backbone, pSB3C5, which was obtained from the iGEM Distribution Kit and was originally cloned with RFP, re-ligating with its original insert rather than the toehold. The team inoculated the colonies that were not producing RFP. Sequencing then confirmed that colony 3 included the correct toehold sequence.

This shows the transformation of the part BBa_K2974700, the BBa_J23100 promoter Toehold eGFP construct, with pSB3C5 under UV light.

Dual Plasmid Transformation of BBa_K2974700 with BBa_K2550001

The dual plasmid transformation of the part BBa_K2974700, the strong promoter and Toehold eGFP construct, in pSB3C5, and part BBa_K2550001, the trigger sequence, in pSB6A1, produced green fluorescent colonies. The low-grade green fluorescence in the colonies indicated a successful binding of the toehold and trigger sequence resulting in the expression of eGFP. There was only a two-day incubation period required by the strains containing the part BBa_K2974100 assembled with BBa_K2550001, possibly due to the relative strength of the strong promoter over the medium promoter.

This is the dual plasmid transformation of the part BBa_K2974700 assembled with pSB3C5 and part BBa_K2550001 assembled with pSB6A1 under UV light.

OPENCELL

Time Optimization

In order to effectively compare OpenCell to a commercial bead homogenizer, Qiagen's DNeasy Plant Pro DNA Extraction protocol had to be optimized for OpenCell. We experimented with different homogenization times to determine the minimum amount of time spent in homogenization that can yield results that are not significantly different from those resulting from longer times spent in homogenization.

Comparison with Commercial Bead Homogenizer

GSU homogenized microalgae with a BioSpec Mini-BeadBeater. No live cells are present.

GSU homogenized microalgae with OpenCell. Live and motile cells are present.

The OpenCell device was sent to GSU's iGem team to compare its effectiveness with GSU's commercial bead homogenizer. In terms of their workflow, GSU iGem determined that OpenCell was more effective than the commercial homogenizer mainly because OpenCell did not kill microalgae cells, while the commercial homogenizer did. OpenCell was also tested against the commercial homogenizer with spinach plants to further determine its effectiveness.

OpenCell's effectiveness with spinach plants was determined first by using Qiagen DNeasy Plant Pro DNA extraction kit and protocol. 20 trials were done, with 10 trials homogenized with OpenCell, and 10 trials homogenized with a BioSpec Mini-BeadBeater. The protocol was followed exactly: samples were homogenized for 2 minutes inside beadbeating tubes included in the kit.

OpenCell was then compared with the BioSpec Mini-BeadBeater by using the team's optimized protocol designed specifically for OpenCell. 20 trials were done, with 10 trials homogenized with OpenCell, and 10 trials homogenized with a BioSpec Mini-BeadBeater. The modifications include: 6 minutes spent in homogenizing rather than 2, homogenizing with 8 3.2mm chrome steel beads within 2 mL tubes rather than the tubes provided by Qiagen, and using 600 μL of lysis buffer rather than 500 μL.

Helminth Homogenization

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.

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.

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 the PCR, protocol for a PCR cleanup was done to purify the amplified DNA. Following the amplification of the DNA, the next step would be to use the HiScribe T7 High Yield RNA Synthesis Kit to transcribe the trigger DNA into RNA in vitro with the use of T7 RNA Polymerase. Lambert iGEM plans to do this step in the future. After converting the resulting DNA to RNA, the single stranded RNA trigger sequence would be introduced to the toehold created by Lambert iGEM and bind to the toehold, causing the hairpin loop of the toehold to unravel and allow for translation and expression of the reporter gene GFP, ultimately demonstrating our work.