iGEM 2023 — Team toronto

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{{subheading("The Lab Calendar")}}
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{{subheading("Wet Lab Notebooks")}}
Download via links below:

[Part 1](%7B%7Bcdn%28%27wetlab-notes/labnotes-part1.pdf%27%29%20%7D%7D)

[Part 2](%7B%7Bcdn%28%27wetlab-notes/labnotes-part2.pdf%27%29%7D%7D)

[Part 3](%7B%7Bcdn%28%27wetlab-notes/labnotes-part3.pdf%27%29%7D%7D)

[Part 4](%7B%7Bcdn%28%27wetlab-notes/labnotes-part4.pdf%27%29%7D%7D)

[Part 5](%7B%7Bcdn%28%27wetlab-notes/labnotes-part5.pdf%27%29%7D%7D)

[Part 6](%7B%7Bcdn%28%27wetlab-notes/labnotes-part6.pdf%27%29%7D%7D)

[Part 7](%7B%7Bcdn%28%27wetlab-notes/labnotes-part7.pdf%27%29%7D%7D)

[Part 8](%7B%7Bcdn%28%27wetlab-notes/labnotes-part8.pdf%27%29%7D%7D)

[Part 9](%7B%7Bcdn%28%27wetlab-notes/labnotes-part9.pdf%27%29%7D%7D)

[Part 10](%7B%7Bcdn%28%27wetlab-notes/labnotes-part10.pdf%27%29%7D%7D)

[Part 11](%7B%7Bcdn%28%27wetlab-notes/labnotes-part11.pdf%27%29%7D%7D)

[Part 13](%7B%7Bcdn%28%27wetlab-notes/labnotes-part131.pdf%27%29%7D%7D)

[Part 14](%7B%7Bcdn%28%27wetlab-notes/labnotes-part14.pdf%27%29%7D%7D)

[Part 15](%7B%7Bcdn%28%27wetlab-notes/labnotes-part15.pdf%27%29%7D%7D)

[Part 16](%7B%7Bcdn%28%27wetlab-notes/labnotes-part16.pdf%27%29%7D%7D)

[Part 17](%7B%7Bcdn%28%27wetlab-notes/labnotes-part17.pdf%27%29%7D%7D)

[Part 18](%7B%7Bcdn%28%27wetlab-notes/labnotes-part18.pdf%27%29%7D%7D)

[Part 19](%7B%7Bcdn%28%27wetlab-notes/labnotes-part19.pdf%27%29%7D%7D)

[Part 20](%7B%7Bcdn%28%27wetlab-notes/labnotes-part20.pdf%27%29%7D%7D)

[Part 21](%7B%7Bcdn%28%27wetlab-notes/labnotes-part21.pdf%27%29%7D%7D)

[Part 22](%7B%7Bcdn%28%27wetlab-notes/labnotes-part22.pdf%27%29%7D%7D)

[Part 23](%7B%7Bcdn%28%27wetlab-notes/labnotes-part23.pdf%27%29%7D%7D)

[Part 24](%7B%7Bcdn%28%27wetlab-notes/labnotes-part24.pdf%27%29%7D%7D)

[Part 26](%7B%7Bcdn%28%27wetlab-notes/labnotes-part26.pdf%27%29%7D%7D)

[Part 27](%7B%7Bcdn%28%27wetlab-notes/labnotes-part27.pdf%27%29%7D%7D)

[Part 28](%7B%7Bcdn%28%27wetlab-notes/labnotes-part28.pdf%27%29%7D%7D)

Missing part numbers are due to oversized pdfs

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Several divisons within our team underwent the thorough and iterative process of engineering design and demonstrated successful results following key takeaways.
In particular, the wet-lab divison was able to showcase this process effectively, during the plasmid curing experiment and Inverse PCR experiment for CRISPR.The team was able to condense the engineering design process into four simple steps: Design, Build, Test, and Learn.
The process with two iterations is described below:**Plasmid Curing Engineering Design Process:**Objective: The primary objective of this project is to eliminate the pETM6 plasmid from the bacterial strain T-B18.**Cycle 1: Temperature Curing**

1. 1. Design: The initial phase of the project involved exploring temperature-based plasmid curing. We hypothesized that subjecting bacterial cells to elevated temperatures would induce stress on the cells, causing them to shed the pETM6 plasmid.
2. 2. Build: To initiate the experiment, a "baseline" culture of the T-B18 bacterial strain was established. The culture was grown under standard conditions, and subsequently, kanamycin was introduced to apply selective pressure.
3. 3. Test: We executed the experimental design by cultivating a portion of the baseline culture at an elevated temperature of 40°C. The objective was to observe whether this increased selective pressure would prompt the bacterial cells to eliminate the pETM6 plasmid. Unfortunately, this attempt was unsuccessful, as the bacterial cells still exhibited resistance to kanamycin.
4. 4. Learn: The initial temperature-based plasmid curing method did not yield the desired outcome. The T-B18 strain retained its resistance to kanamycin, indicating that the pETM6 plasmid was not effectively eliminated through this approach.

**Cycle 2: Antibiotic Curing (Rifampicin)**

1. Design:
In response to the initial failure, we shifted our approach to antibiotic curing using rifampicin. Rifampicin functions by inhibiting bacterial RNA polymerase, which is crucial for transcription. Plasmid DNA replication is dependent on transcription; therefore, inhibiting this process can lead to plasmid loss.2. Build:
The project advanced to the rifampicin sensitivity test. Various dilutions of the T-B18 culture were prepared, each exposed to different concentrations of rifampicin, ranging from 0 ug/mL to 50 ug/mL. This laid the groundwork for subsequent tests.3. Test:
The rifampicin sensitivity test demonstrated significant promise. Under the highest concentration of rifampicin (50 ug/mL), no colonies grew, while the negative control with 0.0 ug/mL of rifampicin exhibited colony growth. Distinct colonies appeared on the plate exposed to 10 ug/mL of rifampicin, prompting us to proceed with a replica plate.4. Learn:
The rifampicin sensitivity test results indicated that T-B18 was sensitive to rifampicin at 50 ug/mL, validating the viability of rifampicin plasmid curing. Subsequent colony PCR, followed by gel electrophoresis of the PCR products, confirmed the loss of the pETM6 plasmid in one of the colonies from the kanamycin replica plate. This marked a significant success in our plasmid curing project, indicating that our intended objective had been achieved using rifampicin.
**Conclusion:**In summary, the engineering design process was executed in a two-cycle approach. While the initial attempt using temperature-based curing was unsuccessful, the shift to rifampicin antibiotic curing led to the successful elimination of the pETM6 plasmid. This iterative approach exemplifies our commitment to refining our methods and achieving our project objectives. Our team will now continue to build upon these findings and explore further applications for this successful plasmid curing approach.

**Inverse PCR Engineering Design Process:**Objective: The primary objective of this project is to construct the pTargetF plasmid with a specific sgRNA sequence for targeted gene knockout, following the two-plasmid CRISPR-Cas9 system.**Cycle 1: Inverse PCR**

1. 1. Design: The initial design was based on the methodology established by Yang et al. (2015) for the knockout of the tpiA gene using the CRISPR-Cas9 system. This design employed a two-plasmid system, where the Cas9 gene and its sgRNA were segregated into the pCas9 and pTarget plasmids, respectively.
{{img(cdn("pictures/ipdesign1.png"), alt = "Fig. 1: Using ChopChop to select top output.")}}
{{img(cdn("pictures/ipdesign2.png"), alt = "Fig. 2: Primer Design on SnapGene.")}}2. 2. Build: To build the pTargetF plasmid, a specific sgRNA sequence was designed and primers, sgRNA\_F\_Old and sgRNA\_R\_Old, were prepared to insert the sgRNA sequence after the gRNA scaffold of the pTargetF plasmid.
{{img(cdn("pictures/ipbuild1.png"), alt = "Table 1: Initial Primer Sequences.")}}3. 3. Test: The experimental setup involved utilizing inverse PCR to amplify the pTargetF plasmid. Phusion polymerase, known for its high fidelity, was used to ensure accurate DNA replication for long amplifications. A specific thermal cycling protocol was employed, and gel electrophoresis was performed to confirm amplification. Unfortunately, primer-dimer issues were encountered, as indicated by the presence of unintended bands on the gel.
{{img(cdn("pictures/iptest1.png"), alt = "Table 2: Initial Thermal Cycling Protocol.")}}4. 4. Learn: The sample shows an obvious primer-dimer band at the bottom of the gel. The presence of primer dimer bands in the gel indicates that some of the primers have annealed to each other and amplified to form unintended products. Possible steps to address primer dimer issues include optimising primer concentrations, template concentration, and adjusting annealing temperatures.
   For primer concentrations, we tried out 10uM to 100uM. For template concentration, we tried repeated plasmid miniprep to ensure the nanodrop result is of higher concentration. For annealing temperature, we tried out from a range of 48C to 57C. None of the experiments yielded positive results.
{{img(cdn("pictures/iplearn1.png"), alt = "Fig. 3: Gel Results showing clear Primer Dimer.")}}
**Cycle 2: Inverse PCR (Revised Design)**

1. Design: Following the primer-dimer issues encountered in Cycle 1, the design was revisited. Input was sought from a colleague who had experience with the same CRISPR-Cas9 system and had achieved positive results. Based on this input, the primer design for inverse PCR was revised.2. Build: The revised primers, sgRNA\_F and sgRNA\_R, were designed to be used in inverse PCR for inserting the sgRNA sequence into pTargetF plasmid.{{img(cdn("pictures/ipbuild2.png"), alt = "Table 3: Revised Primer Sequences.")}}
3. Test: In the second cycle, a standardized PCR protocol provided by NEB was followed. Gel electrophoresis was conducted to confirm the successful amplification. Notably, the gel electrophoresis results showed a distinct band at approximately 2000bp, matching the expected size for the amplification of the pTargetF plasmid. Additionally, the nanodrop results indicated an acceptable DNA concentration and purity, facilitating future transformation steps.{{img(cdn("pictures/iptest2.png"), alt = "Table 4: Revised Thermocycling Protocol.")}}
4. Learn: The revised primer design in Cycle 2 effectively resolved the primer-dimer issues encountered in Cycle 1. The standardized PCR protocol and the revised primer design led to the successful amplification of the pTargetF plasmid, as confirmed by gel electrophoresis. Nanodrop result also indicates an acceptable concentration and DNA purity to enable future transformation steps. This achievement allows for the mass production of pTargetF\_tpiA as a stock.{{img(cdn("pictures/iplearn2.png"), alt = "Fig. 4: Gel Results showing successful Inverse PCR; from left to right: Ladder, PCR reaction 1, PCR reaction 2, positive primer design control, Phusion master mix control.")}}
{{img(cdn("pictures/iplearn3.png"), alt = "Table 5: Recorded Nanodrop concentrations.")}}
**Conclusion:**In summary, the project to construct the pTargetF plasmid using inverse PCR underwent two cycles of the engineering design process. The initial cycle faced challenges related to primer-dimer formation, which were addressed by revising the primer design in the second cycle. This iterative approach allowed for the successful construction of the pTargetF plasmid, meeting the project's objective. The ability to mass-produce the pTargetF\_tpiA plasmid as a stock is a significant milestone in this endeavour.
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{{ subheading("Experimental Background")}}

{{ subsubheading("Overarching Rationale") }}

Landfill gas emissions are a growing problem, releasing tonnes of
methane and carbon dioxide into the atmosphere and surrounding
environment. This is of particular concern in Toronto, with there being a
large number of Indigenous communities located in close proximity to
landfill sites, and therefore receiving the brunt of the pollution. This
year, iGEM Toronto has been working to remedy the issue of landfill gas
pollution, by means of using synthetic biology to transform bacteria into
functional methylotrophs. Our experimentation over the past five months
has centred on improving methanol and other one-carbon compound (C1)
assimilation and reducing glucose dependency of our bacterial strains,
with specific attention to E. coli as our model organism. To achieve our
goal of shifting the metabolisms of bacteria from glucose to methanol
consumption, we have worked to optimize the metabolism of C1 substrates
such as formate or methanol by means of gene knock-outs, cell culturing
and transformation, quantification of metabolite uptake, and adaptive
laboratory evolution (ALE).

{{ subsubheading("Improving upon Existing Work") }}

To begin our experimentation, we needed a baseline to build upon; this
came in the form of Har et al.’s (2021) work on the ALE of E. coli strain
BW25113 ΔfrmA that was transformed with the goal of improved methanol
derived carbon metabolism, resulting in creation of E. coli T-B18. This
strain was specifically engineered to use C1 feedstock to synthesize
amino acids, and was determined to be an ideal model organism due to its
strong methylotrophy and ease of replicability. We were further inspired
by iGEM Wageningen University & Research’s 2021 project that focused on
the biofiltration of methane from cattle farms in the Netherlands by
means of engineering bacterial chassis including a different strain of
E. coli to improve methane oxidation. Learning from these two sources,
iGEM Toronto has bridged the gaps between the creation of methylotrophic
strains of E. coli and the identified need for a reduction in C1 emissions
by applying the optimization of C1 substrate uptake to the context of
common pollutants from landfills: methanol and formate. We have furthered
the development of methylotrophy by means of conducting genetic knock
outs and replacements within E. coli T-B18.

{{ subsubheading("Gene Choice") }}

Determining what genes should be manipulated within our baseline strain
began with the identification of our pathway of interest and its key
players; we focused on the reductive glycine pathway, RUMP cycle, and
glycolysis, and identified the genes tpiA, frdA, and mdh as targets of
engineering. The gene tpiA, as discussed in more depth below, codes for
triosephosphate isomerase, and serves to ensure that the RUMP cycle is
reversible - for the purposes of increasing this pathway in the
direction of methanol assimilation, we conducted a CRISPR-Cas9 knockout.
The gene frdA codes for one component of the Fumarate Reductase enzyme
complex, which was identified through Flux Balance Analysis (FBA) by
our Dry Lab team as being a strong candidate for knockout that would
improve methanol assimilation. Lastly, the overexpressed gene mdh for
methanol dehydrogenase was replaced as described below to optimize the
catalysis of methanol to formaldehyde, which is the first reaction in
the RUMP pathway.

{{ subsubheading("Quantification Choice/Troubleshooting & ALE Rationale") }}

To identify the level of methanol assimilation, we carried out steps for
quantifying the metabolites of our baseline and transformed metabolites.
To carry this out, we turned to using High Performance Liquid
Chromatography (HPLC) as a means of profiling the amount of methanol
present in cell culture before and after transformation. A secondary
quantification protocol using gas chromatography was also considered.
There were many considerations for our method of quantification,
including processing capacity, ease of quantification for volatile
metabolites, and specificity for cell cultures. To further demonstrate
an improvement in C1 substrate utilization, our experimental protocol
calls for ALE as a means to showcase the long-term shift of the bacterial
chassis towards complete methylotrophy.

{{ subheading ("Plasmid curing of pETM6 from T-B18")}}

T-B18 (E. coli BW25113 ΔfrmA)[1] has the plasmid pETM6\_Ptrc\_BsMdh\_BmHps\_BmPhi[2]. T-B18 has Kanamycin (KAN) resistance inserted into the genome and pETM6 has Ampicillin (AMP) resistance. Because future CRISPR experiments require transforming 2 extra plasmids (pCas9 and pTargetF) into T-B18, we want to cure out pETM6 to reduce plasmid burden when doing CRISPR. Also, the mdh substitution requires T-B18 without pETM6\_Ptrc\_BsMdh\_BmHps\_BmPhi. Hence we will first cure pETM6 and create a glycerol stock of T-B18 ΔpETM6.

{{img(cdn("pictures/wl-plasmidcuring.png"),alt="Fig. 1: Plasmid Curing Workflow")}}

{{ subheading("Replacement of B. m Mdh with C. n Mdh in pETM6 ") }}

Mdh, encoding methanol dehydrogenase, is the first reaction in the RuMP cycle. This enzyme is responsible for catalyzing the conversion of methanol to formaldehyde, as it needs to be introduced heterogeneously into the engineered chassis, E. coli. According to multiple previous studies on engineered methylotrophs, mdh is considered as the bottleneck reaction of the pathway. In the paper that describes the T-B 18 strain, the overexpressed mdh gene is derived from B. stearothermophilus, as it has been proven to perform better than the conventionally used B. Methanolicus mdh in vivo in E. coli. However, in an endeavor to improve the conversion rate of methanol, we identified an mdh sequence that has superior Km parameters to increase the enzyme's specificity for methanol. This is also supported by the paper that generated the mdh-containing plasmid, which claims that more effective mdh models, such as the one generated by Liao et al. in 2016, exist. Liao et al. describes a high-throughput screening of mutagenized mdh models generated by directed evolution in an E. coli host. A superior mdh variant, CT4-1, is identified to possess high specificity towards methanol and high conversion efficiency, with a Km value of 21.6 ± 1.5 mM and a Km/Kcat of 9.3 M−1 s−1. This led the team to hypothesize that by substituting the original mdh expression vector with C. necator CT4-1 mdh, would result in an improvement in the overall methanol assimilation in our engineered strain.

{{img(cdn("pictures/wl-mdh.png"),alt="Fig. 2: Mdh Replacement Workflow")}}

{{ subheading(" CRISPR-Cas9 Knockout") }}

There are multiple genetic targets that could be subjected to knockout to increase the metabolic flux of methanol assimilation in Rump pathway. Specifically, we identified one target from Vorholt et al. 2020, that deletion of the triosephosphate isomerase (tpiA) gene leads to the increased theoretical flux of the Rump pathway. TpiA catalyzes the glycolysis pathway's reversible conversion between dihydroxyacetone phosphate (DHAP) & D-glyceraldehyde 3-phosphate (GAP). Deleting the tpiA gene would impede GAP derived from methanol, in which case it could only be generated from pyruvate. This increases the flux of DHAP formation from methanol, resulting in higher incorporation of methanol into Rump cycle intermediates.
As expected, the ΔtpiA strain demonstrated methanol-dependent growth, agreeing to their in silico studies. The metabolic burden on the synthetic pathway is found to be low as well, with 6.5 % of the biomass derived from methanol. Furthermore, the computation predicts that as much as 40% of the methanol could be incorporated by Rump cycle with tpiA deletion.
The team deems tpiA would be a good Knockout candidate for T-B18 strain for our case since numerous conditions from both papers are transferable. For example, they use the same starting strain E. coli BW25113 ΔfrmA, the engineering approaches include episomal overexpression of the essential Rump cycle genes (mdh, hps & phi), and both of the paper focused on improving the upstream methanol assimilation capability as their primary goals.

{{img(cdn("pictures/wl-tpia-ko-fin.png"),alt="Fig. 3: Impact of tpiA KO")}}

We decided to use the CRISPR-Cas9 gene editing system to achieve tpiA knockout in our T-B18 E.coli as it is efficient, well established, and has high specificity compared to other methods.
We will be using two-plasmid systems: pCas9 + pTarget for CRISPR Cas9 System. pCas contains cas9 gene with a native promoter, an arabinose-inducible sgRNA guiding Cas9 to the pMB1 replicon of pTarget, the lambda-Red recombination system to improve the editing efficiency, and the temperature-sensitive replication repA101(Ts) for self-curing. We have checked that the origin of replication of pCas9 is compatible with the pTarget, and the pTarget series contains the targeting sgRNA. The Donor DNA is purchased from IDT as a linear DNA fragment.

The CRISPR-Cas9 genome editing designs for knockout of tpiA is based on a study conducted by Sheng Yang and colleagues in 2015 (Yang et al., 2015), necessitates a two-plasmid system. In the two-plasmid system, the Cas9 gene and its corresponding sgRNA, directing it to the tpiA region of E.Coli, were segregated into the pCas9 plasmid and a pTarget plasmid, respectively. The repair template donor DNA is provided as a separate fragment.
The plasmid in which carries cas9 endonuclease gene was replaced from the original paper in order to adjust to the antibiotic resistance in our E.Coli strain. While the pTargetF plasmid consists of the sgRNA scaffold sequence, the N20 sequence of tpiA, and multiple restriction sites. The N20 sequences (CAAGTGATCATTCAGTACGG) of tpiA is incorporated in the backbone of pTargetF (Addgene #62226) using inverse PCR with primers (forward primer: gttttagagctagaaatagc, reverse primer: gctatttctagctctaaaacCCGTACTGAATGATCACTTGactagtattata). The repair donor template is 1000bp synthesized DNA that has 500bp homologous to both upstream and downstream of the tpiA region in the genome.

{{img(cdn("pictures/wl-tpiakoworkflow.png"),alt="Fig. 4: tpiA Knockout Experiment Workflow")}}

{{ subheading("Quantification") }}

In order to verify the efficacy of the methylotrophic traits of the engineering strain, the team conducted quantification using High-Performance Liquid Chromatography (HPLC). HPLC is an analytical technique that separates compounds in a chemical mixture based on pressure-driven flow. HPLC operations consist of a mobile phase and a column-packed stationary phase; various compounds in the mobile phase interact differently with the stationary phase and are hence separated due to elution in the column. The sample, in this case, the growth media, is placed in the mobile phase. Following the separation, a detector measures the relative intensity of a compound in the mobile phase and plots it accordingly on a chromatogram. The chromatogram is the resulting visual from this method, and for our experiment, it provides information on the methanol concentration present in the media post-growth. An HPLC measurement is taken at time zero (t0) prior to culture growth and then sampled consistently over the phases of the cell growth curve. A successful experiment would indicate a steady decline in the methanol concentration in the media which indicates that the bacterial strain assimilated the methanol, utilizing it as a metabolite and therefore demonstrating methylotrophic tendencies.

{{img(cdn("pictures/wl-hplcworkflow.png"),alt="Fig. 5: HPLC Experiment Workflow")}}

{{ subheading("Adaptive Lab Evolution") }}

Our engineered-T-B18 strain, which is a methylotrophic E. coli strain derived from the wildtype strain T-B18 described by Antioniewics et al. 2021, has the potential to improve its methanol assimilation capability. To achieve this, we will use adaptive laboratory evolution (ALE) to apply evolutionary pressure to enhance a specific trait. The original paper that generated T-B18 focused on removing amino acid auxotrophy, while methanol was used to boost growth. However, our goal is to create a strain that relies on methanol alone, with minimal or no dependence on heterotrophic carbon sources such as glucose.

Our ALE procedure will involve subjecting the strain to two stress factors in the culture media: higher methanol concentration and lower glucose concentration. We hypothesized that this will enable the strain to adapt and increase its methanol utilization efficiency, while decreasing its glucose intake dependency.

ALE is commonly conducted either through serial passaging or using a chemostat. Due to time and resource constraints, our team has opted for serial passaging in favor of its straightforwardness. Serial passing involves manually transferring culture to new media with gradually increasing levels of stress. In our case, given the growth rate of our strain, culture was transferred every two days to new media where the concentration of glucose decreased for each subsequent passage. The most common approach involves culturing and passaging of strains with flasks. This allows for larger volume, but is manually challenging due to the requirement of a large number of flasks given our need to pass multiple strains. The approach we decided on was to use 96 well culture plates with a multichannel pipette for transferring. This method would be more convenient to conduct, and allow us to run multiple populations in parallel, but it risks a smaller volume for ALE.

As per the original paper, the serial transfer procedure is conducted as follows.Their engineered strain grew in rich media for 36 days with continuous passing. The strain was then subjected to minimal media (M9) containing threonine and supplemented with 250mM of methanol. The stressor for their experiment, threonine, was decreased in concentration from 10mM to 2mM in a period of 70 days.

We adapted their methodology to our experiment. Our stressor will be decreasing glucose for each subsequent passage. Starting with regular M9 media with 4g/L of glucose, it was decreased by 0.36g/L each passage for a total of 10 passages to a final glucose concentration of 0.4g/L.
To identify the strain that grew fastest, colonies were isolated through plating and cultured overnight in the same conditions. An OD600 reading was taken to identify the colonies that grew fastest. Sequencing was done on the fastest growing colony to identify the mutations that took place.

{{img(cdn("pictures/wl-ale.png"),alt="Fig.6 : ALE Experiment Workflow")}}

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