Guelph iGEM

Results

Plasmid Preparation and Confirmation

Background

What is MoClo?
MoClo is a modular system for assembly of genetic elements based on the use of type IIS restriction enzymes and golden gate cloning (Lee et al.,). In this method, genetic elements can be considered “modular parts,” which may be combined in golden gate assembly (GGA) to create larger genetic constructs. For example, the three parts below are assembled to form out lead sensor.

S. cerevisiae cells
Figure 1. Promotor, aptamer, and reporter gene are assembled via complementary overhangs to form a composite part.

Why MoClo?
MoClo allows us to easily swap modules to create different genetic circuits. For instance, the GFP Dropout part in our entry vectors can be interchanged for any part(s) designed to have complimentary overhangs when cut with the same type IIS restriction enzyme. In this specific example, GFP is substituted with the three parts that make up our lead sensor. However, any genetic parts with the same overhangs and cut sites can be used in this substitution, meaning infinite modules can be made from swapping just one gene.

S. cerevisiae cells
Figure 2: GFP is swapped out of pENTRY for 3 modular parts with complimentary overhangs. In this case, pTEF2, Pb7S, and GFP (-AUG) are used.

How does MoClo work?
MoClo is a step wise process, involving the hierarchical assembly of basic modules into multi-gene plasmids.
Step 1: Creating entry vectors (pENTRY)
Basic parts containing BsaI cut sites are assembled in a 1 pot reaction. Digestion using this type IIS restriction enzyme leaves behind complimentary overhangs, which can only ligate together in a pre-defined order.

S. cerevisiae cells
Figure 3. pENTRY is assembled from basic parts. A GFP part with internal BsaI cut sites is placed in between the left connector (ConL) and right connector (ConR) for future green-white colony screening.

Each pENTRY plasmid uses a different set of ConL/ ConR parts, which may be cut by BsmBI to create pre-defined overhangs for ligation of multiple pENTRYs.

Gel A

A)

Gel B

B)

Gel C

C)

Figure 4. Three pENTRY plasmids. For simplicity, only GFP and its flanking ConL/ ConR parts are shown. Complimentary connectors are displayed in the same color.

Step 2: Creating Transcriptional Units
GFP is replaced by modules of interest via its internal BsaI cut sites. Any module designed with complimentary overhangs can thus be inserted into pENTRY to form transcriptional units.

S. cerevisiae cells
Figure 5. Two unique transcriptional units are assembled from the same pENTRY backbone.

Step 3: Transcriptional units are combined to form a multi-gene plasmid
Transcriptional units from step 2 are digested with BsmB1 in GGA, allowing them to ligate via their connectors.

S. cerevisiae cells
Figure 6. Transcriptional units are assembled into a multi-gene plasmid. Matching colors represent complimentary CON parts. This allows for ligation of modules of interest, while preventing pENTRY backbone repetition.

Results

Creating Entry Vectors (pENTRY)
Basic parts were assembled into entry vectors using GGA and BsaI.

S. cerevisiae cells
Figure 1. SnapGene assembly of the five entry vectors (pENTRY) later used as transcriptional unit backbones. A to D represent pENTRY-ConLS-R1, pENTRY-ConLS-RE, pENTRY-ConL1-RE, pENTRY-ConL1-R2, and pENTRY-ConL2-RE, respectively.

Plasmids were extracted from green-white screened E. coli DH5α transformants, digested with NcoI, and analyzed by agarose gel electrophoresis to confirm size in base pairs (bp). Expected size for all pENTRY constructs is 4747 bp.

S. cerevisiae cells
Figure 2. 1.0% agarose gel electrophoresis of NcoI digested pENTRY plasmids using the NEB 1kb+ ladder as a molecular weight standard. Lanes 2 to 6 contain pEntryConLS-R1, pEntryConLS-RE, pEntryConL1-RE, pEntryConL1-R2, and pEntryConL2-RE, respectively. Bands align with expected length of 4747 bp.

Detection Testing Overview

Background

Basic Principle
The detection method is based on the study, Artificial protein-responsive riboswitches upregulate non-AUG initiation via ribosomal stalling by Horie et al. (2020). This study shows that the yeast ribosome pre-initiation complex can be induced to translate from a non-cognate start codon (NCSC) such as CUG instead of AUG by a riboswitch. The engineered riboswitch is designed to interfere with the 48S ribosome pre-initiation complex (PIC) scanning for AUG start codon on the mRNA, via formation of RNA secondary structures produced from aptamer-ligand interactions2. The system exploits an increase of aptamer stability when the a ligand is bound to it, thereby physically preventing the ribosome initiation complex from scanning the mRNA and inducing it to initiate translation at the first non-cognate start codon closest to the ribosomal binding site.

S. cerevisiae cells
Figure 1. Translation occurs from non-cognate start codon when ligand is bound to aptamer (riboswitch), producing a fusion protein

Applications for Detection of Lead

S. cerevisiae cells
Figure 2. Production of GFP (-AUG) by a lead-specific aptamer. Binding of ligand to aptamer confers stability and forces the PIC to recognize the NCSC. 60S ribosomal subunit is recruited, and translation occurs. Fusion GFP is produced and fluorescence is observed.

We use the principle above to develop a system to sense Pb2+ using lead binding aptamer as the riboswitch and a GFP gene lacking a start codon as the reporter. GFP fusion protein, translated from the non-cognate start codon (NCSC) will only occur when lead is present.

S. cerevisiae cells
Figure 3. In the absence of lead, the aptamer is not stable enough to prevent PIC scanning, and translation fails to initiate.

In the absence of lead, no translation occurs, and no GFP is produced. Samples do not fluoresce.

Results

Creating Transcriptional Units for Riboswitch Testing
Three transcriptional units were created for riboswitch testing; two test units, and a positive control. The positive control had sequence errors that that precluded it from providing useable data, and thus will be excluded from analysis. Test plasmids contain in the following order: pTEF2, a strong promotor; Pb7S or Pb14S, an aptamer; and GFP without an AUG start codon. The positive control contained pTEF2 and GFP with an AUG start codon. GFP in pENTRY-ConLS-ConR1 was swapped for respective modules of interest during assembly.

S. cerevisiae cells
Figure 4. SnapGene assembly of pPb7S (A) and pPb14S (B) transcriptional units used to compare aptamer function. pPb7S-Test contains Pb7S aptamer, with a Kd constant of 1.60 ± 0.16 µM. pPb14S-Test contains Pb14S aptamer, with a Kd constant of 0.76 ± 0.18 µM. Aptamers selected from Yang et al., 2018.

Constructs were transformed into E. coli DH5α. Four white colonies per construct were subjected to colony PCR. Primers targeted at ConLS and ConR1 were used to amplify the 1700 bp inserted modules of interest. Amplicons were analyzed using gel electrophoresis.

S. cerevisiae cells
Figure 5. 1.0% agarose gel electrophoresis of pPb7S-Test and pPb14S-Test inserts amplified via colony PCR. NEB 1kb+ ladder used as a molecular weight standard. Yellow arrows indicate pPb7S-Test amplicons. Orange arrows indicate pPb14S-Test amplicons. All amplicons have an expected size of 1700bp. Underlined samples aligned with expected size and were selected for whole plasmid sequencing.

Samples pPb7S-Test-C, pPb7S-Test-D, pPb14S-Test-B, and pPb14S-Test-D were sequenced at the AAC Genomics Facility with Oxford Nanopore PromethION 24. pPb7S-Test-C, pPb7S-Test-D, and pPb14S-Test-B returned positive for correct sequence.

Site-Directed-Mutagenesis of Riboswitch Transcriptional Units

Riboswitch transcriptional unit assembly deviated from the three-step MoClo workflow described in plasmid preparation background. Saccharomyces cerevisiae was transformed with pPb7S-Test-C, pPb7S-Test-D, and pPb14S-Test-B, and the positive control. The control colonies did not fluoresce under UV light and instead were white. The control module should constitutively express GFP, resulting in green colonies. Inspection of the sequence revealed an ATG start codon upstream the GFP start codon, causing a frameshift. Further inspection of pPb7S-Test and pPb14S-Test revealed 2 errors:
  1. An ATG start codon upstream of the non-cognate start codon (NCSC), causing a frameshift
  2. NCSC was not in frame with GFP, causing a second frameshift

Site-directed mutagenesis (SDM) was used with custom primer sets for pPb7S-Test and pPb14S-Test, followed by Dpn1digest to remove template DNA. Primers could not be ordered for positive control, thus it was discarded. Primers were designed to introduce 2 mutations to pPb7S-Test and pPb14S-Test:
  1. Change the unwanted ATG start codon to ACG
  2. Insert bases downstream of the NCSC to correct the reading frame.
Gel electrophoresis of SDM-PCR products was used to confirm amplification, followed by Oxford Nanopore PromethION 24 sequencing of plasmid extracted from E. coli DH5α.
S. cerevisiae cells
Figure 6. 1.0% agarose gel electrophoresis of pPb7S-Test-C (1CQ), and pPb14S-Test-B (2BQ) SDM PCR products. NEB 1kb+ ladder used as a molecular weight standard. Reactions used 10 ng of template DNA and Q5 polymerase. PCR was followed up by Dpn1 digest with 1ul (1CQ1, 1BQ1) or 2ul (2BQ1, 2BQ2) of enzyme. Gel confirmed amplification, but is not specific enough to reveal if mutation was successfully introduced. Expected size of all amplicons is 5.3 kb.
Sequencing returned positive for 1CQ2 and 1BQ2.

96 Well Plate Assay
pPb7S, pPb14S and wild-type yeast at OD600 1.0 were inoculated into YPD with 0.2 ug/ul of Lead Nitrate. Samples shook at 200 cpm at 30 degrees Celsius, for 4 hours total on an MiBS plate reader. An excitation wave length of 395 nm was used. No usable reads were collected, due to an overflow error, where fluorescence detected exceeded the amount that could be read.
S. cerevisiae cells
Figure 1. Triplicates of yeast transformed with pPb7S in column 1, pPb14S in column 2, and wild-type in column 3. Fluorescence was too high for an accurate read.

Discussion

Strong fluorescence is shown in both pPb7S and pPb14S yeast. Data could not be collected because fluorescence exceeded the MiBS plate reader limits. Background florescence observed in wild-type yeast cannot be normalized without absorbance readings. This assay will therefore be conducted again using more dilute samples, blanks containing only YPD, and a positive control module. See future directions for more information. These preliminary results are inconclusive, but promising.

References

Reference:

  1. Horie F, Saito T, Kato S, et al. (2020) Artificial protein-responsive riboswitches upregulate non-AUG initiation via ribosomal stalling. ACS Synth Biol. . doi: 10.1021/acssynbio.0c00206
  2. Querido J, Sokabe M, Kraatz S, et al. (2020) Structure of a human 48S translational initiation complex. Science. doi: 10.1126/science.aba4904
  3. Yang C, Li H, Gao T, et al. (2018) Selection of DNA aptamers for the Development of Light-Up Biosensor to Detect Pb (II). Sens. Actuator B-Chem. doi: 10.1016/j.snb.2017.07.068

Memory Testing Overview

Background

Basic Principle
Our riboswitch-aptamer system should provide a fluorescent read-out due to GFP production when Pb2+ is present. Our team wanted to explore the possibility of creating a memory system to allow for permanent and irreversible expression of reporter genes to an initial transient exposure of lead (Pb2+). Our design was based on a serine integrase that can irreversibly flip ON a state switchable promoter (SSP), to allow for transcription of a reporter gene.

The memory system is reliant on two components: (1) the ability of serine integrase to facilitate site-specific recombination, and (2) a state switchable promotor (SSP) flanked by attachment sites (attB and attP) recognized by an integrase enzyme. The promotor is initially in an OFF configuration since its orientation is opposite to the desired direction of transcription. This means no genes downstream the promotor will be expressed. When serine integrase is present, it will catalyze the permanent inversion of the SSP into an ON-configuration, allowing for the constitutive transcription of linked downstream genes. After the SSP has been flipped to the correct orientation, chimerc attL and attR sequences will flank the SSP. The SSP cannot be flipped back to the “OFF” position by the integrase due to the chimeric att sequences and the absence of recombination directionality factor (RDF) accessory proteins3.

S. cerevisiae cells
Figure 1. pTEF2 promotes constitutive transcription of integrase. The SSP is in an off state, and downstream response genes are not transcribed. Integrase is folded into a mature protein, which acts upon attP and attB sites flanking the SSP. Integrase mediated inversion forms attL and attR sites that flank the SSP, now in its forward orientation. Response genes are constitutively transcribed by the SSP.

Application in Genetic Circuit
Using the lead aptamer system described previously, we envisaged that serine integrase can be placed in frame with the non-cognate start codon instead of GFP. Therefore, in the presence of Pb2+, integrase is produced, and it will that will catalyze the inversion of the SSP to its forward orientation. Genes for the sequestration of lead can be linked to the SSP. Once the SSP has been irreversibly flipped to its ON state by integrase, sequestration genes will be constitutively expressed, meaning the yeast will “remember” their exposure. Additionally, this system allows for the amplification and persistent expression of downstream genes in all progeny cells once a transient signal has been detected.

S. cerevisiae cells
Figure 2. Expression of integrase (-AUG) depends on ligand-aptamer binding. In the presence of lead, integrase is produced from the NCSC. This fusion protein acts upon attP and attB sites to irreversibly flip an SSP into its forward orientation. Genes linked to the SSP are constitutively expressed.

Since integrase have not been demonstrated to function in yeast, we first decided to test its utility by creating a reporter system shown below. PTEF2 is used to constantly express integrase. Directly downstream is a LacZ gene placed in a reverse (backwards) conformation and flanked by attP/ attB recognition sites. This form of LacZ cannot be transcribed by pTEF2, because its conformation places ATG out of frame. If the expression and function of integrase is successful, LacZ will be flipped into an ON-configuration (forward) at its attP/ attB sites, and ATG will be in frame with pTEF2. This active LacZ gene is expressed, and B-galactosidase is produced. B-galactosidase can cleave o-nitrophenyl-β-D-galactopyranoside (ONPG), visible as a color change at 420 nm. The absorbance values for pIntegrase-Test will be compared against positive control pLacZ-Positive, and 2 negative controls: pLacZ-negative, and wild-type yeast. A significant difference in absorption between pIntegrase-Test and pLacZ-Negative indicates successful integrase-mediated inversion of LacZ.

S. cerevisiae cells
Figure 3. Integrase acts upon attB and attP flanking LacZ. Chimeric attL and attR sites are formed. LacZ is placed in frame with pTEF2, causing B-Galactosidase expression. In ONPG, this is visualized as a yellow color change.

Results

Creating Transcriptional Units for Integrase Testing
Three transcriptional units were created for integrase testing; pLacZ-Positive, a positive control; pLacZ-Negative, a negative control; and pIntegrase-Test, a test plasmid. The pLacZ-Positive module contained a pTEF2 promotor, and LacZ. The pLacZ-Negative module contained, in order, a pTEF2 promotor, a short spacer sequence, and inverted (backwards) LacZ flanked by attP and attB sites. The pIntegrase-Test module contained, in order, a pTEF2 promotor, ΦBT1 serine integrase, and inverted (backwards) LacZ flanked by attP and attB sites. GFP in pENTRY-ConLS-ConR1 was swapped for respective modules of interest during assembly.

S. cerevisiae cells
Figure 5. SnapGene Assembly of pIntegrase-Test (A), pLacZ-Negative (B), and pLacZ-Positive (C).

Plasmids were extracted from 3-4 white E. coli DH5α transformants, and digested with PstI for size-analysis by gel electrophoresis.

Gel A

A)

Gel B

B)

Gel C

C)

Figure 6. 1.0% agarose gel electrophoresis of PstI-digested transcriptional units, using the NEB 1 kb+ ladder as a marker for molecular weight. (A) Integrase-Test has an expected size of 9520 bp. (B) LacZ-Negative has an expected size of 7753 bp. (C) LacZ-Positive has an expected size of 7759 bp. Underlined samples were selected for whole-plasmid sequencing.

Samples pIntegrase-Test-B, pIntegrase-Test-C, pLacZ-Negative-B, pLacZ-Negative-C, pLacZ-Positive-C, and pLacZ-Positive-D were sequenced at the AAC Genomics Facility with Oxford Nanopore PromethION 24. pIntegrase-Test-C, pLacZ-Negative-C, pLacZ-Positive-C, and pLacZ-Positive-D returned positive for correct sequence.

Due to time constraints, the iGEM Guelph team was only able to collect experimental data for the function and efficiency of integrase. Visit Future Directions for next steps.

Ortho-Nitrophenyl-β-Galactoside (ONPG) Assay
Yeast was transformed with pIntegrase-Test-C, pLacZ-negative-C, and pLacZ positive-C samples selected from sequencing results.
A colorimetric Ortho-Nitrophenyl-β-Galactoside (ONPG) assay was conducted on all 3 modules, in addition to Wild-type BY4741 for a secondary negative control. Cells were grown in synthetic complete medium lacking uracil (SC-URA) for 72 hours at 37C with 220rpm of shaking, centrifuged and then lysed with glass beads in 250 μL of breaking buffer containing phenylmethylsulphonyl fluoride (PMSF). 100 μL of cell extract was added to 900 μL of Z-buffer and incubated at 28C for 5 minutes. 200 μL of ONPG stock solution was then added to initiate the reaction and begin the assay. For more information, please visit Experiments.
S. cerevisiae cells
Figure 1. Data analysis of the results collected from the ONPG assay of Saccharomyces cerevisiae normalized to yeast cell density (OD600). In yeast transformed with pLacZNegative, β-galactosidase activity is not significantly different to wild-type Saccharomyces cerevisiae. Yeast transformed with pIntegraseTest show significant β-galactosidase activity when compared to wild-type S. cerevisiae determined by student’s t-test, although activity is much lower than the positive control (pLacZPositive). Significant differences in β-galactosidase relative to yeast lacking an entry vector are designated by asterisk (student’s t-test, two-tails; * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001).

Discussion

The data indicates that ΦBT1 serine integrase is indeed functional within S. cerevisiae. However, further testing is required to fully characterize and optimize ΦBT1 efficiency. See future directions for our next steps in testing ΦBT1 serine integrase.

References

Reference:

  1. Essington E, Vezeau G, Cetnar D, Grandinette E, Bell T, Salis H, et al. An autonomous microbial sensor enables long-term detection of TNT explosive in natural soil. Nature Communications. 2024 Dec 2;15:10471. doi: 10.1038/s41467-024-54866-y.
  2. Merrick CA, Olorunniji FJ, McPherson A, et al. Serine integrases: advancing synthetic biology. ACS Synth Biol. 2018;7(2):299–310. doi:10.1021/acssynbio.7b00308.

Downstream Gene Expression

Background

Goal of Gene Expression
The final module of our regulatory genetic circuit consists of the genes expressed by the state-switchable promoter in the ON state, after ΦBT1 serine integrase has translated in cells that have detected lead (Pb2+).
Work completed by Sun et al.1 proved the successful engineering of Saccharomyces cerevisiae into a plant-like hyperaccumulator of various metals. We aim to express an operon-like series of genes in S. cerevisiae to achieve the hyperaccumulation of lead (Pb2+).1

What Genes Will We Express?
The genes selected for expression aim to enable S. cerevisiae to uptake and sequester Pb2+ at a greater capacity. The three genes selected for expression are as follows:

  • Divalent Metal Transporter 1(DMT1)
  • γ-Glutamylcysteine Synthetase (GSH1)
  • Yeast Cadmium Factor 1 (YCF1)

The DMT1 gene produces a metal transporter that enables the increased uptake of extracellular Pb2+. Subsequently, the expression of GSH1 increases intracellular glutathione (GSH) levels, which bind the toxic free Pb2+ ions and form a Pb–GSH complex.4 This complex is then recognized by YCF1, an ATP-binding cassette (ABC) transporter which facilitates the transport of Pb–GSH complexes into vacuoles.5

S. cerevisiae cells
Figure 1. DMT1, GSH1, and YCF1 function together to transport and sequester lead in yeast.

How Will an Operon-Like Series of Genes Be Constructed?
To control the transcription of multiple Pb2+ uptake and sequestration genes (as mentioned previously) under one promoter, we plan on linking each gene via a viral “self-cleaving” 2A peptide6. The viral 2A peptide self-separation is caused by the translation of a conserved D-V-E-X-N-P-G-P motif6. When the final glycine (G) and proline (P) of the motif (mentioned previously) is translated, the ribosome stalls and fails to form a peptide bond between the two amino acids6. This effectively separates any protein products before and after the motif, allowing for the translation of multiple genes with a single mRNA transcript6. See future directions for information on testing downstream gene expression.

Basic Principle
The detection module uses a genetic construct where lead ions bind to an RNA aptamer, activating translation of GFP. Fluorescence intensity therefore correlates with the presence and concentration of lead, allowing for a measurable visual output.

Applications for Detection of Lead
This system could be applied to inexpensive and portable biosensors for lead detection in drinking water, soil, and industrial waste. With optimization, the fluorescence-based output could be adapted for real-time monitoring or on-site environmental testing.

References

Reference:

  1. Sun GL, Reynolds EE, Belcher AM. Designing yeast as plant-like hyperaccumulators for heavy metals. Nature Communications. 2019 Nov 8;10(1):5080. doi: 10.1038/s41467-019-13093-6.
  2. Ren Y, Lin Q, Berro J. 2A peptide from ERBV-1 efficiently separates endogenous protein domains in the fission yeast Schizosaccharomyces pombe. microPublication Biology. 2023 Sep 11;2023:000941. doi: 10.17912/micropub.biology.000941./li>
  3. Bannon DI, Portnoy ME, Olivi L, Lees PSJ, Culotta VC, Bressler JP. Uptake of lead and iron by divalent metal transporter 1 in yeast and mammalian cells. Biochemical and Biophysical Research Communications. 2002 Jul 26;295(4):978-984. doi: 10.1016/S0006-291X(02)00756-8
  4. Ask M, Mapelli V, Höck H, Olsson L, Bettiga M. Engineering glutathione biosynthesis of Saccharomyces cerevisiae increases robustness to inhibitors in pretreated lignocellulosic materials. Microbial Cell Factories. 2013;12:87. doi: 10.1186/1475-2859-12-87
  5. Khandelwal NK, Millan CR, Zangari SI, Avila S, Williams D, Thaker TM, Tomasiak TM. The structural basis for regulation of the glutathione transporter Ycf1 by regulatory domain phosphorylation. Nature Communications. 2022 Mar 11;13(1):1278. doi: 10.1038/s41467-022-28811-w
  6. Souza-Moreira TM, Navarrete C, Chen X, Zanelli CF, Valentini SR, Furlan M, Nielsen J, Krivoruchko A. Screening of 2A peptides for polycistronic gene expression in yeast. FEMS Yeast Research. 2018 Aug 1;18(5):foy036. doi: 10.1093/femsyr/foy036