iGEM 2025 — Team imperial


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{% block title %}Engineering{% endblock %}
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On This Page
[Colony PCR](#colony-pcr)
[Hoc](#hoc)
[CRISPR](#crispr)
[dPMI](#dpmi)
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# Engineering

# Engineering Cycle

Throughout our design and experimentation phase, we iterated through various DBTL cycles within the different engineering targets.
We compiled a list of the major engineering cycles that influenced our project direction and have listed them below.

# Colony PCR of *E. coli* GF Plasmids

## Cycle One

**Design:** Primers were initially designed to span across both the backbone and the insert.

**Build:** Golden Gate assembly of the plasmid constructs were performed using a cycling protocol.

**Test:** Ran colony PCR and visualised the results using gel electrophoresis. The gel returned empty.

**Learn:** As successful insertion of the plasmid backbone was required for the colony PCR to produce any band, we were unable to troubleshoot this result very effectively. Specifically, we were unable to conclude whether the negative gel was due to a problem in the colony PCR protocol or a problem with assembly and transformation.

## Cycle Two

**Design:** We ordered new primers that bind only to the backbone, where transformation of an empty or correct backbone plasmid would show a smaller band in the gel. As the primers bind the region which flanks the gene insert, successful transformants can be identified through a larger sized amplicon.

**Build:** We repeated the colony PCR protocol from the previous cycle with some modifications. Namely, we increased the initial holding time from 1 minute to 5 minutes in order to facilitate better initial denaturation of plasmid DNA.

**Test:** We performed gel electrophoresis to visualise the results. We noted the absence of large amplicons in all samples, indicating a failed assembly.

**Learn:** We were able to identify whether the issue leading to our low number of successful transformants was an assembly or a transformation issue. We found that the plasmid assembly was unsuccessful for our growth factor plasmids.

## Cycle Three

**Design:** To address a potential issue with enzyme compatibility, we designed a protocol that would use only NEB reagents to ensure the buffer would be compatible with all relevant enzymes. NEB’s BsaI-HFv2 and BbsI-HF were used along with rCutSmart buffer supplemented with 1 mM ATP.

**Build:** Golden Gate assembly of our growth factor constructs was performed using an overnight isothermal protocol.

**Test:** We transformed *E. coli* and performed colony PCR on the transformants to assess correct assembly.

**Learn:** There was increased transformation efficiency and we observed fewer colonies on the control (empty) assembly transformation, indicating the plasmid backbone may have been enzymatically linearised during assembly. However, the colony PCR gel had distinct bands, but all the plasmids were negative for the correct assembly. We concluded that there must be some problem with the assembly, and after checking our experimental plan, realised that we were not accounting for the length of the plasmid backbone the parts were shipped in during our Golden Gate assembly calculations, which affected the fragment volumes required for high-efficiency assembly.

## Cycle Four

**Design:** We re-calculated the Golden Gate assembly protocol to account for the plasmid backbone length.

**Build:** We re-ran Golden Gate assembly with the updated reagent volumes.

**Test:** We transformed *E. coli* and performed colony PCR on the transformants to assess correct assembly.

**Learn:** We saw bands which indicated distinct larger amplicons on some of the colonies, indicating that some assemblies had worked. Namely, EGF, HGF, and IGF-Rho all showed successful assembly. Regarding the unsuccessful assemblies, we theorised that we had run out of usable FGF plasmid by this point, and that the concentration of mCherry plasmid was lower than what we measured by Nanodrop quantification. We chose to transform TOP10 *E. coli* with mCherry plasmid and inoculate an overnight to amplify.

# Hoc

## Cycle

**Design:** During our initial literature review, multiple scholarly articles indicated a truncated *Hoc1* gene led to a more permeable cell membrane. We investigated the potential biochemical mechanism for this difference.

**Build:** Protein sequences for the wild-type and truncated Hoc1 were generated and their structures were first predicted *in silico* using AlphaFold 3.

**Test:** To assess the potential loss of activity in the truncated enzyme, we performed a structural analysis of the active site pocket in PyMOL. Previous studies identified several conserved residues involved in donor substrate binding (T143, D225, D277, Q300, V336) and in stabilisation (D179, R213). These residues are highly conserved across yeast and mould species and are essential for enzymatic function[1].

**Learn:** Our analysis revealed that one of the active site pockets is severely affected by truncation, with most of it missing. Among the conserved residues, Q300 is lost due to the frameshift, likely impairing the enzyme’s substrate binding ability and stability, thereby potentially reducing enzyme activity.

![Superimposed structures of truncated and original Hoc1](https://static.igem.wiki/teams/5916/assets/pymoling.avif)

**Figure 1.** Superimposed image of truncated and original Hoc1 structure. (A) Surface representation of the enzyme’s active site, illustrating the reduced binding pocket caused by the frameshift-induced truncation. (B) Zoomed view of the active site pocket, highlighting the conserved residues responsible for substrate stabilisation and binding. The residue Q300 is absent in the truncated structure. Structures were predicted using AlphaFold 3.

---

**[1]** E. T. R. Kelly, D. Rodionov, B. Sleno, P. A. Romero, and A. M. Berghuis, ‘Crystal structure of the fungal mannosyltransferase Och1 reveals active site primed for N-glycan binding’, *PLoS One*, vol. 20, no. 7, p. e0329259, July 2025, doi: 10.1371/journal.pone.0329259.

# CRISPR

## Cycle One

**Design:** Designed oligonucleotides that would assemble sgRNAs targeting the *Hoc1* locus. Gene fragments were designed to have high-fidelity Golden Gate overhangs for assembly.

**Build:** DNA fragments were assembled using overlap extension PCR.

**Test:** Construct sequence length was verified by gel electrophoresis.

**Learn:** Plasmid construction was successful. We moved on to Golden Gate assembly.

## Cycle Two

**Design:** Golden Gate assembly was designed to construct the final sgRNA–Cas9 plasmid.

**Build:** Golden Gate assembly with BbsI enzyme was performed, after which the plasmids were transformed into chemically competent TOP10 *E. coli*.

**Test:** Observed colonies on corresponding antibiotic selection plates and sequence verified assemblies.

**Learn:** *E. coli* transformation was successful, and we were able to move on to electrocompetent *K. phaffii* transformation.

## Cycle Three

**Design:** We designed a protocol and prepared electrocompetent *K. phaffii* for electroporation. We amplified a pair of homologous recombination templates that would allow for the downregulation and/or repair of the *Hoc1* gene.

**Build:** We transformed *K. phaffii* with sgRNA plasmid and HR template, a plasmid containing the downregulated and/or repaired *Hoc1* gene for *K. phaffii*.

**Test:** Plated transformants onto Hygromycin and Hygromycin + Nourseothricin (NRS) combination plates to select for transformants containing the Cas9 plasmid. Colonies which survived were then plated onto NRS plates to select for the gene insert.

**Learn:** Colonies grew on Hygromycin plates but were unable to proliferate on Nourseothricin plates. This indicates that while the Cas9–sgRNA plasmids were correctly transformed, we had insufficient concentrations of the HR templates causing the integration of the cassette to fail.

## Cycle Four

**Design:** We adjusted the protocol to increase the concentration of DNA present during *K. phaffii* electroporation.

**Build:** We repeated *K. phaffii* electroporation protocol using HR template DNA we gathered and concentrated down from five minipreps.

**Test:** Plated transformants onto Hygromycin and Hygromycin + Nourseothricin (NRS) combination plates to select for transformants containing the Cas9 plasmid. We saw no survival of colonies throughout the plates.

**Learn:** As we used a different electroporator model and conducted the protocol without direct supervision, we considered possible issues deriving from the machinery. Upon reviewing the settings of the electroporator, we identified that the time constant varied from our initial run, which likely decreased *K. phaffii* cell viability. This was unfortunately something that could not have been controlled on the electroporator we used for this experiment.

## Cycle Five

**Design:** We continued to use the higher plasmid DNA concentration and conducted the electroporation protocol under the correct settings.

**Build:** We repeated *K. phaffii* electroporation under the same conditions as the first attempt.

**Test:** Observed colonies which were able to grow on the combination Hygromycin and Nourseothricin selection plates, indicating potentially successful transformants.

**Learn:** *Hoc1* expression being controlled by the PIS1 promoter may be viable in *K. phaffii*. Further testing is required to confirm these modifications have taken place. If successful, testing of promoter repression using zinc would be useful to characterise this behaviour.

# dPMI

## Cycle One

**Design:** Designed corresponding sgRNAs targeting the *PMI* locus. Gene fragments were designed to have high-fidelity Golden Gate overhangs for assembly.

**Build:** sgRNAs were assembled by overlap extension PCR. Plasmids were assembled using Golden Gate assembly and transformed into TOP10 *E. coli*.

**Test:** Observed colonies on corresponding antibiotic selection plates and assemblies were sequence verified.

**Learn:** *E. coli* transformation was successful and we yielded 4 out of 5 sequence-perfect clones. We determined that we could use these sgRNA plasmids to transform *K. phaffii*.

## Cycle Two

**Design:** From our previous experiment, we determined that we could transform *K. phaffii* with PMI-targeting sgRNAs. We designed a template for the homologous recombination of a downregulated copy of PMI in place of the original gene, purifying and linearising it to obtain enough potential DNA for two transformations.

**Build:** We ran *K. phaffii* electroporation.

**Test:** Plated transformants onto Hygromycin and Hygromycin + Nourseothricin (NRS) combination plates to select for transformants containing the Cas9 plasmid. Colonies that survived on combination plates were re-plated on Nourseothricin plates to remove the burdensome Cas9 plasmid.

**Learn:** Colonies were able to grow on Hygromycin plates but were unable to proliferate on NRS plates. This indicates that empty Cas9 plasmids were transformed, where insufficient plasmid DNA concentration was the likely cause for the unsuccessful transformants.

## Cycle Three

**Design:** We adjusted the protocol to increase the concentration of DNA present during *K. phaffii* electroporation.

**Build:** We ran *K. phaffii* electroporation.

**Test:** Plated transformants onto Hygromycin and Hygromycin + Nourseothricin (NRS) combination plates to select for transformants containing the Cas9 plasmid. There were no colonies which survived.

**Learn:** As we used a different electroporator model and conducted the protocol without direct supervision, we considered possible issues deriving from the machinery. Upon reviewing the settings of the electroporator, we identified that the time constant varied from our initial run, which likely decreased *K. phaffii* cell viability.

## Cycle Four

**Design:** We continued to use the higher plasmid DNA concentration and conducted the electroporation protocol under the correct settings.

**Build:** We ran *K. phaffii* electroporation.

**Test:** Observed colonies which were able to grow on the combination Hygromycin and Nourseothricin selection plates, indicating successful transformants.

**Learn:** We need to run colony PCR in the future to confirm our modifications were successful.

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## What Should this Page Contain?

---

Provide comprehensive, step-by-step protocols for all experiments conducted. Include a list of materials, reagents, and equipment used. Specify concentrations, volumes, incubation times, temperatures, and other critical parameters. Document any modifications or optimizations made to standard protocols.

Explain the purpose of each experiment and its relevance to your project goals. Describe the experimental design, including controls and replicates. Provide a clear rationale for the chosen methods and approaches.

Document any troubleshooting steps taken and optimizations made during the experimental process. Share any lessons learned that could benefit future teams.

## Inspirations

---

* [2024 Heidelberg](https://2024.igem.wiki/heidelberg/experiments)
* [2024 Marburg](https://2024.igem.wiki/marburg/experiments)
* [2024 EPFL](https://2024.igem.wiki/epfl/experiments)
* [2023 Estonia-TUIT](https://2023.igem.wiki/estonia-tuit/experiments)

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On This Page
[CRISPR](#crispr)
[Growth Factors](#growth-factors)
[Bioreactor](#bioreactor)
[BioKernel Validation Experiment](#biokernel)
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# RESULTS

# Introduction

Throughout our work in the lab this summer, we were able to establish the potential viability of mutant
*K. phaffii* strains not characterised in literature with regards to our CRISPR modifications and our
growth factor constructs.

In particular, we demonstrated the potential viability of both the
**Hoc1 downregulation–repair** modification and the **PMI downregulation** modification,
both of which would be novel to iGEM if future testing confirms their success.

# CRISPR

We successfully demonstrated the transformation of electrocompetent *K. phaffii* with sgRNA plasmids quite early in our experiments.

We successfully assembled our sgRNA constructs as verified by gel electrophoresis. We expected a single band around 250 bp from the assembled construct, which we saw on all lanes with DNA added on the gel. We consequently gel purified this and were able to isolate a substantial amount of sgRNA for use in assembly.

![Gel electrophoresis of sgRNA constructs](https://static.igem.wiki/teams/5916/wetlab/results1.avif)

Gel electrophoresis showing assembled sgRNA constructs (~250 bp).

We successfully transformed *E. coli* with sgRNA plasmids to verify sequence fidelity via sequencing.
Below: PMI 2324 sgRNA transformed into BB3cH backbone and amplified using TOP10 *E. coli*.

![PMI 2324 sgRNA transformation](https://static.igem.wiki/teams/5916/wetlab/results2.avif)

*Figure 2. Transformed *E. coli* colonies containing PMI 2324 sgRNA.*

Six out of ten sgRNA plasmids sequenced returned as 100% correct clones, confirming successful assembly.

![Test digest verification of sgRNA plasmids](https://static.igem.wiki/teams/5916/wetlab/results3.avif)

*Figure 3. Test digest verification of sgRNA plasmid assemblies.*

We were unfortunately unable to fully confirm the success of our transformation protocol. We were unable to isolate a substantial mass of HR repair templates throughout our time in the lab, even when performing up to nine minipreps of 3 mL overnight cultures per template.

Each time we increased the number of minipreps and each time we failed to isolate enough plasmid to run a substantial number of transformations, we were reminded of the frustrating variability within synthetic biology and the limitations of yield on our experiments even when using various methods to concentrate our DNA.

Despite these setbacks, we saw some promising signs during our experiments. Our final round of transformation saw colonies grow on YNB–NRS plates — a sign that the HR repair template had been taken up into the *K. phaffii*.
We were unable to verify whether the templates were integrated or not by colony PCR.
We also experienced some filamentous fungi contamination in this round, but it seems that we were able to isolate uncontaminated colonies successfully.

![Colonies grown on YNB–NRS plates](https://static.igem.wiki/teams/5916/wetlab/results4.avif)

Colonies on YNB–NRS plates showing possible *K. phaffii* transformants.

# Growth Factors

Our growth factor experiments were characterised by a substantial amount of troubleshooting.

After correcting concentrations, we achieved successful assembly as verified by test digest.

![Test digest showing successful Golden Gate assemblies](https://static.igem.wiki/teams/5916/wetlab/results5.avif)

*Figure 5. Test digest showing successful Golden Gate assemblies.*

Following transformation into *K. phaffii*, we obtained Zeocin-resistant colonies,
though initial plates showed fungal contamination.

![IGF_Rho transformants with contamination](https://static.igem.wiki/teams/5916/wetlab/results6.avif)

*Figure 6. IGF–Rho transformants showing fungal contamination on YNB–Zeo plates.*

Re-streaking successfully isolated uncontaminated *K. phaffii* colonies.

![Restreaked IGF_Rho transformants](https://static.igem.wiki/teams/5916/wetlab/results7.avif)

Re-streaked IGF–Rho transformants showing successful isolation of *K. phaffii*.

Though we were unable to verify the correct insertion or assembly of our construct in *K. phaffii*,
we have reason to believe that we have strains capable of expressing recombinant human growth factors
when induced with methanol.

Though sequence verification is pending, these strains likely express recombinant human growth factors upon methanol induction.

# Bioreactor

Due to the lack of growth of our growth factor transformants, we were unable to isolate a successfully modified strain for characterisation work in a bioreactor.
We were, however, able to characterise a baseline run of the bioreactor with the trHoc1 Type strain PN-2 and record extremely helpful data concerning optimal run conditions and nutrient flow rates for culturing of
*K. phaffii* at scales larger than the benchtop.

We started with 25 mL of *K. phaffii* PN-2 culture in approximately 560 mL of buffered basal salts medium.
We observed the bioreactor foam up immediately, likely attributed to the high final cell density of our starting culture, and added some antifoam.

![Bioreactor foaming during K. phaffii PN-2 run](https://static.igem.wiki/teams/5916/wetlab/results8.avif)

Bioreactor foaming observed during *K. phaffii* PN-2 baseline run.

After approximately 55 hours, we were able to harvest roughly 500 mL of liquid culture.
We observed that a non-trivial amount of media had been lost to evaporation.

We pelleted each tube using a centrifuge at maximum speed for 10 minutes before filtering the supernatant.
We were able to isolate the wet cell pellet and the supernatant, a portion of which was filtered through 0.2 µm filters.
These filters almost immediately clogged, indicating the potential presence of large proteins and/or sugars remaining in the supernatant.

We were unfortunately unable to quantify and characterise these large molecules in time for the wiki freeze.
We are presently continuing further investigation of this to present at the jamboree.

# BioKernel Validation Experiment

We established an efficient workflow for the assembly and cloning of vectors carrying our enzymes of interest.

After a successful assembly and transformation of Stage 1 parts, we performed a colony PCR to assess the efficiency of parts insertion with this kit.

Of 76 colonies screened, 50 were positive, demonstrating the relatively high efficiency of correct transformation using this kit and its constituent plasmids.

![Colony PCR of EcoFlex assembly kit](https://static.igem.wiki/teams/5916/wetlab/results9.avif)

Colony PCR of EcoFlex assembly demonstrating 50/76 successful insertions.

We were unfortunately unable to clone the plasmids into the Stage 3 destination vector in time for the wiki freeze.
Despite this, we are continuing this work and hope to accomplish this soon for the characterisation of the astaxanthin pathway in *E. coli*.

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