Engineering

Design #1:

Our initial goal was to design an expression construct for EPG-Nanoluciferase, a simple single-CDS construct that has been proven to work effectively. For protein overexpression, we utilized the T7 expression system, which is known to function well in BL21(DE3) cells, facilitating high levels of target protein production.

Build #1:

To create the plasmids, we used components from a distribution kit and a synthesized part, NanoLuc_EPG, synthesized from Twist Bioscience. We began by transforming these plasmids into commercially available DH5α cells to prepare for subsequent Golden Gate assembly. However, due to antibiotic selection issues, we were unable to isolate pure clones carrying the desired plasmid. In a subsequent attempt, most parts were successfully cloned, but the promoter sequences failed to clone properly. We suspect that specific interactions between the plasmid and host cells contributed to the low success rate in transformation and cloning.

Additionally, our attempts to extract DNA via Miniprep were largely unsuccessful, affecting our ability to obtain high-quality plasmids for further assembly.

Test #1:

Due to the failure in obtaining the desired constructs, no downstream testing could be performed in this round.

Learn #1:

Several issues were identified throughout the process:

• Transformation Issues:

  The initial transformations were not successful. Troubleshooting revealed a few critical factors:

  • Antibiotics for selection plates should be mixed into the media only when the temperature is below 55°C.
  • The heat shock during transformation should be precisely 30 seconds.
  • For creating chemically competent cells, the TSS method proved to be the most effective. Additionally, all glassware must be thoroughly cleaned and autoclaved with water to avoid detergent contamination.
• DNA Quality Issues:

  While PCR amplification of the DNA itself did not present any problems, there was significant variation in the number of colonies formed across different transformation attempts, pointing to inconsistencies in transformation efficiency.

• Promoter Plasmid Issues:

  The plasmid containing the promoter sequences was not transformed successfully, indicating a potential issue with either the plasmid construct or the transformation conditions.

• M13 Primer Issues:

  PCR amplification using M13 primers for the pOpenV3 plasmid, which contains the open gene constructs, was unsuccessful when using Q5 polymerase. Despite testing four different annealing temperatures (53°C, 55°C, 57°C, and 58°C), no correct-sized PCR product was obtained, indicating a potential primer binding issue or incompatibility with the chosen conditions.

Design #2:

We made a series of adjustments to our protocol for the Golden Gate assembly. These changes included swapping out the promoter and origin of replication and modifying the steps for plasmid extraction, PCR purification, Golden Gate assembly, and transformation. To improve DNA quality and yield, we PCR-amplified all parts directly from the distribution kit rather than relying on miniprep plasmids. Additionally, we switched from chemical transformation to electroporation to enhance transformation efficiency.

The promoters we tested are as follows:

• T7LacO (BBa_J435350) was replaced with:
• pTac (BBa_J435360)
• J23100 (BBa_J23100)

Build #2:

We successfully constructed the plasmids and confirmed their sequences. However, sequencing revealed some issues: one construct contained mutations in the promoter region, and another was missing both the promoter and ribosome binding site (RBS).

Test #2:

To test the construct's functionality, we initially placed a magnet beneath the plate to activate the EPG construct. According to the EPG protocol, we expected a clear signal of activation. However, we observed a suspicious increase in the activity of the TEV constructs even in plates without magnetic activation, suggesting an unintended rise in background activity.

Initially, we suspected that this activity could be caused by the innate magnetism of the plate reader. To confirm this, we repeated the experiment using a strong external electromagnet but did not observe any significant activation during the initial trials.

Learn #2:

• Mutations in Kit Components:

  The distribution kit may have contained mutations, leading to altered promoter sequences and issues with overhangs that affected cloning efficiency.

• Activation Protocol Challenges:

  The current activation setup is suboptimal for a 96-well plate format. While the magnetic field is homogeneously distributed for single samples, it is insufficient for uniformly activating all wells in a larger plate.

• Challenges with EPG Characterization:

  The EPG construct appears to have magnetic sensitivity, but our current setup may not be suitable to accurately characterize its response. The magnetic activation may either lack sufficient sensitivity or not produce a homogeneous effect, making it difficult to differentiate signal from noise. Further optimization is needed to ensure clear and consistent activation across experiments.

Design #3:

Our initial design aimed to identify a viable protein target that could incorporate EPG (Electromagnetic Perceptive Protein) into other proteins. The objective was to control protein activity, thereby translating a magnetic signal into one that can be recognized by other biological systems. After exploring several options, we selected two promising candidates for fusion: EPG-TEV and EPG-LacI.

Build #3:

We retrieved the sequences for both proteins and designed a series of fusion proteins by connecting EPG to the target proteins through various linker sequences and at different fusion sites. This process yielded multiple constructs for each fusion type.

Test #3:

We then used AlphaFold3 for structural prediction to assess the feasibility of the fusion proteins and the effects of different insertion sites. In total, we screened 40 fusion protein constructs.

See the model page for more information

Learn #3:

We discovered that the EPG-LacI fusion is unlikely to be functional. The LacI protein domain is responsive to lactose through a hinge region, and for the fusion protein to function effectively, the EPG sequence with its signal peptide would need to be inserted into an amorphous loop within the hinge domain. However, our structural predictions revealed minimal conformational diversity in the LacI-EPG fusion when compared to the EPG-TEV fusion. This lack of flexibility suggests that the EPG-LacI fusion may not be effective.

In contrast, the EPG-TEV fusion demonstrated better modularity, with more conformational diversity among constructs and greater consensus across different constructs. This suggests that the EPG-TEV fusion is a more viable option with a higher potential for functionality.

Design #4:

We aimed to test approximately 20 constructs of the EPG-TEV fusion protein, allowing us to screen various linker lengths and combinations. To facilitate this, we designed a Golden Gate assembly system with custom overhangs to enable easy swapping of linkers during assembly.

To monitor TEV activity within cells, we also designed a reporter plasmid encoding a fusion protein of sfGFP and REACh2, with an internal TEV cleavage site. REACh2 is a non-fluorescent variant of YFP that absorbs the fluorescence from sfGFP through Förster Resonance Energy Transfer (FRET). This design ensures that the intact fusion protein is non-fluorescent, while cleavage by TEV produces green fluorescence, allowing for easy monitoring of TEV activity.

In addition to the main constructs, we also designed positive and negative control plasmids for accurate assessment.

Build #4:

We assembled the constructs using Golden Gate cloning; however, colony PCR did not yield any results, suggesting unsuccessful cloning or issues with the assembly process.

Test #4:

Due to the failure in obtaining the desired constructs, no further tests were conducted. At this point, we faced significant time constraints, limiting additional troubleshooting and optimization efforts.

Design #5:

To create a homogeneous magnetic field and facilitate easy adjustments, we designed an activation box capable of eliminating magnetic field (MF) interference and providing precise activation. To enhance the intensity of the fluorescence signal and minimize readout noise, we increased the concentration of the substrate by fivefold. A more comprehensive series of tests was conducted to further characterize EPG behavior under different conditions.

Build #5:

The activation box was built using 3D printing. More details can be found in the hardware documentation.

Test #5:

We tested the EPG constructs at four different magnetic field intensities:

• Strong: 24 mT
• Medium: 12 mT
• Weak: 6 mT
• Control (No Activation): less than 1 mT

We also conducted tests without pre-reading the chemiluminescence levels to isolate the effects of MF activation on the signal. Additionally, a "double activation" test was performed, where a second round of MF activation and subsequent readout was carried out after the initial activation and reading. All these experiments were conducted at the strong magnetic field level.

Learn #5:

The results indicated that EPG activation might actually reduce EPG activity, which contradicts previously published data. A possible reason for this discrepancy is the lack of real-time measurement capability, which prevented us from monitoring changes in fluorescence as the MF was applied. Although we developed a custom hardware solution to address this issue, it lacked sufficient sensitivity and was hampered by excessive noise. Unfortunately, due to time constraints, we were unable to conduct further testing or make improvements to the hardware setup.

See the model page for more information.

Throughout our engineering cycles, we focused on designing, building, and testing EPG-Nanoluc fusion protein constructs for controlled magnetic activation. Despite initial challenges in cloning and assembly, we optimized our design and identified EPG-TEV as a promising candidate. We managed to construct EPG-TEV, but it didn't come out successfully. To improve fluorescence signal detection, we increased substrate concentration and developed a custom activation box to provide precise magnetic fields. Additional hardware have been made, but the sensitivity issues have become significant.

Testing showed reductions in EPG-Nanoluc activity upon magnetic activation, which is not similar to existing data and was likely due to limitations in real-time measurement and hardware sensitivity. However, there is a possibility that the construct with the specific linker setup would change the pattern of activation of EPG. These cycles provided insights into the behavior of EPG-Nanoluc, informing future work on optimizing activation and detection systems.

Design #1

To implement thermal pathways in S. cerevisiae, we made use of native heat-sensitive transcription factors. This meant that we only needed to design one plasmid, the reporter plasmid containing a fluorescent protein under the control of a heat-sensitive promoter. Two promoters were used for this: HSP26 and pTDH3. The promoter of the HSP26 locus is upregulated under heat shock, while the pTDH3 promoter is downregulated in heat stress.

Plasmid	Part	Source	Notes
HSF-dependent fluorescent protein expression	Bacterial origin	Distribution kit	BBa_J435282 (pWV01-EColiOrigin)
	Yeast Origin	Distribution kit	BBa_J435290 (Sc2micron_YeastOrigin)
	Yeast selection marker	Distribution kit	BBa_J435287 (ScKanR-marker_YeastMarker)
	Connector	Distribution kit	BBa_J435232 (AConL-start, left-connector)
	HSP26 promoter	Yeast genome	PCRed up to introduce BsaI cut sites and appropriate overhangs
	mTagBFP	Distribution kit	BBa_K592100 (mTagBFP)
	Terminator	Distribution kit	BBa_J435228 (Sc-tENO1_Terminator)
	Connector	Distribution kit	BBa_J435270 (AConR-end, right-connector)
	Bridge	Distribution kit	BBa_J435254 (OYC-bridge-AGAC-CGAA)
	Transfer origin	Distribution kit	BBa_J435286 (oriT_TransferOrigin)
	Bacterial selection marker	Distribution kit	BBa_J435256 (OYC-CamR_EColiSelection)
HSF-dependent fluorescent protein expression	Bacterial origin	Distribution kit	BBa_J435282 (pWV01-EColiOrigin)
	Yeast Origin	Distribution kit	BBa_J435290 (Sc2micron_YeastOrigin)
	Yeast selection marker	Distribution kit	BBa_J435287 (ScKanR-marker_YeastMarker)
	Connector	Distribution kit	BBa_J435232 (AConL-start, left-connector)
	pTDH3 promoter	Distribution kit	BBa_J435209 (Sc-pTDH3_TempOYCPromoter)
	mTagBFP	Distribution kit	BBa_K592100 (mTagBFP)
	Terminator	Distribution kit	BBa_J435228 (Sc-tENO1_Terminator)
	Connector	Distribution kit	BBa_J435270 (AConR-end, right-connector)
	Bridge	Distribution kit	BBa_J435254 (OYC-bridge-AGAC-CGAA)
	Transfer origin	Distribution kit	BBa_J435286 (oriT_TransferOrigin)
	Bacterial selection marker	Distribution kit	BBa_J435256 (OYC-CamR_EColiSelection)

Build #1

We aimed to construct these by Golden gate using the parts listed in the table below; a mixture of synthesised and from the distribution kit. Unfortunately, we had issues with the purification of various parts from the kit. This was done in parallel to the E. coli thermal pathways, see below for more details on the issues and how we tried to solve them. Since we spent a lot of time troubleshooting this, we decided to focus solely on E. coli for thermal pathways going forwards.

Design #1

We initially designed a system with two plasmids (figure 11): one expressing TlpA or TcI under the Tac promoter, and the other expressing a fluorescent protein under the control of TlpA or TcI. It was important that these two plasmids had different replication origins to reduce the burden on the replication machinery, and also that they have different resistance markers to allow selection.

Plasmid	Part	Source	Notes
TlpA/TcI gene expression	ChlR and CloDF13 ori backbone	From instructor	PCRed up to introduce BsaI cut sites and appropriate overhangs
	Tac promoter	Distribution kit	BBa_J435360 (AB_pTac)
	RBS	Distribution kit	BBa_J435374 (BC_B0032)
	TlpA/TcI gene	Synthesised	Ordered as a double-stranded gene fragment from Twist
	T7 terminator	Distribution kit	BBa_J435361 (EF_T7Term)
TlpA-dependent eYFP expression	KanR, pUC ori backbone	Distribution kit	BBa_J435330 (high copy (pUC) ori/KanR)
	TlpA promoter	Synthesised	Ordered as two single stranded oligos, then annealed together
	RBS	Distribution kit	BBa_J435374 (BC_B0032)
	eYFP	Distribution kit	BBa_E0030 (eYFP)
	T7 terminator	Distribution kit	BBa_J435361 (EF_T7Term)
TcI-dependent eYFP expression	KanR, pUC ori backbone	Distribution kit	BBa_J435330 (high copy (pUC) ori/KanR)
	TcI promoter	Distribution kit	BBa_R0051 (Cell_Comm_Promoters_R0051)
	RBS	Distribution kit	BBa_J435374 (BC_B0032)
	eYFP	Distribution kit	BBa_E0030 (eYFP)
	T7 terminator	Distribution kit	BBa_J435361 (EF_T7Term)

Build #1

Test #1

The testing in this cycle was to troubleshoot our transformation and miniprep protocols. For transformations, we tested two different methods - chemical transformation and electroporation. To optimise each of these, we modified various parameters such as recovery time and medium following heat shock/electroporation. While these helped us to increase our transformation efficiency, there seemed to be some plasmids which were just less efficient at transformation, as they repeatedly failed when using the same protocols as others.

For minipreps, we tried two different kits left behind by the previous year's iGEM team. When neither of these worked well, we contacted the companies of these kits and they kindly sent us new reagents. We also tried different parameters, such as varying the OD at which cultures were miniprepped. However, there did not seem to be a significant trend (figure 13). Similar to transformations, there were some parts that frequently had issues.

To aid in our troubleshooting process, we held a meeting with NEB and went through our protocols and troubleshooting process. There was not a clear conclusion reached as the protocols and subsequent adjustments we followed seemed comprehensive, thus leading them to suggest that the issue may lie with the reagents or quality of DNA.

Learn #1

As we lost a lot of time troubleshooting these techniques, we decided to switch to two alternatives. One of these (see cycles 3/4) was to order standard primers that annealed to the backbones of the kit parts, allowing us to amplify up the region we needed. While this was fast and easy, it came with the risk of mutations being introduced into the parts we used. Since the parts we were unable to purify were relatively short (promoter and terminator), we decided that the chance of this was low, particularly as we were using the high fidelity Q5 polymerase.

The other approach we attempted (see cycle 2) was to outsource the synthesis of the plasmids instead. This had the added benefit of allowing us to have both transcriptional units on one plasmid, reducing the burden on the cell. In addition, in the interest of time, we decided to focus solely on E. coli for thermal pathways going forwards.

Design and Build #2

This plasmid contains both of the transcriptional units described in Design #1, with some changes in each unit: (1) the terminator of one was changed to the B0015 double terminator to reduce repetition within the sequence; (2) both ribosome binding sites, because the one we were using before contained a hairpin sequence; (3) codon optimisation of the TlpA/TcI genes and the eYFP genes, to reduce sequence complexity. The units are oriented in opposing directions to reduce transcription issues. The backbone used is the standard IDT pUCIDT (Amp) Golden Gate, which contains the high copy pUC origin and an ampicillin resistance marker. It is also designed to be compatible with golden gate by not containing any of the recognition sites for common Golden Gate enzymes.

This plasmid was entirely ordered as a synthetic construct and chemically transformed into DH5alpha and BL21 cells. These transformations were seen to be successful as cells were able to grow on antibiotic resistant plates.

Test #2

No significant fluorescence seen in hot plate testing or plate reader tests.

Learn #2

While inducible promoters are good for reducing burden on cells, it makes it difficult to pinpoint where things are going wrong - especially without access to an SDS-PAGE or some other method of validating expression of our transcription factor. In addition, we realised that there should be more space between the two promoters to reduce cross-effects of transcription factor binding.

Design #3

Similar to design #1, design #3 is a two plasmid system using parts mostly from the distribution kit. The key changes are to the promoter and terminator that we were unable to purify - the Tac promoter was replaced by a constitutive Anderson promoter (BBa_J23119), while the terminator was switched out for double terminator B0015. We chose the BBa_J23119 promoter because the characterisation on the registry suggested a higher rate of transcription compared to the other tested promoters. We also switched from the eYFP promoter to mRFP1 because we wanted to visualise the fluorescence more easily against the yellow autofluorescence of the LB agar.

Plasmid	Part	Source	Notes
TcI gene expression (1p V1 and V2)	ChlR and CloDF13 ori backbone	From instructor	PCRed up to introduce BsaI cut sites and appropriate overhangs
	Anderson promoter	Distribution kit	BBa_J23119
	RBS	Distribution kit	BBa_J435374 (BC_B0032)
	TcI gene	Synthesised	Ordered as a double-stranded gene fragment from Twist
	Double terminator	Distribution kit	BBa_J428092 (BBa_B0015)
TcI-dependent mRFP1 expression (2p V1 and V2)	KanR, pUC ori backbone	Distribution kit	BBa_J435330 (high copy (pUC) ori/KanR)
	TcI-dependent promoter	Distribution kit	BBa_R0051 (Cell_Comm_Promoters_BBa_R0051)
	RBS	Distribution kit	BBa_J435374 (BC_B0032)
	mRFP1	Distribution kit	BBa_J428036 (mRFP1)
	Double terminator	Distribution kit	BBa_J428092 (BBa_B0015)

Build #3

The promoter and terminator were extracted from the kit by PCR, rather than transformation and miniprep. The plasmids were assembled by golden gate, and two types of transformations were performed. One involved each golden gate reaction being chemically transformed into DH5alpha, as is standard. In the interest of time, we also electroporated both reactions into BL21 at once and plated onto a double antibiotic plate.

Both sets of transformations were successful as colonies grew on their respective single or double antibiotic plates. 1p and 2p V1 cells were heat shocked for 1min, and V2 heat shocked for 30s but both contained the same plasmids. BL21 cells were plated onto plates: α and β with no difference between the cells on each plate. As the BL21 double plasmid cells grew, we proceeded with thermal testing. However, following a colony PCR, we found that the 1p Golden Gate was unsuccessful and some parts were missing (figure 18).

Test #3

The primers we had initially designed for 1p colony PCR were unable to amplify at all. Since the cells were able to grow on double antibiotic plates, we decided to start testing while waiting for new primers for colony PCR. Interestingly, we saw some promising results, despite eventually discovering that the 1p plasmid was missing some parts of the transcriptional unit (figure 18 left).

We first tested the BL21 on the hot plate for 5 hours. At the beginning, the cells were white and remained white after 5 hours. Our positive control was the β2 pink colony as it was pink even at room temperature. Next, we put the plate in a 43℃ incubator for 1 hour. After growing overnight at room temperature, the colonies remained white. After growth at room temperature for another day, the colonies had a slight pink colour in the centre.

Following this test, we wanted to see if expression of mRFP1 could be increased by increasing the incubation time. After reading RDFZ-China 2018 iGEM team's wiki page, we decided to incubate different colonies off the same plate for 24h.
6 colonies from each plate (α and β) were inoculated in liquid as well as plated on solid double antibiotic plates over the weekend at room temperature. After 65h, the colour of the cells were mostly white with some samples either with a pink centre or were fully pink (see table below).

After 24h, the 37℃ and 42℃ plates had grown a lot of colonies. A control plate as also incubated at room temperature, however after 24h, there were no colonies grown. Therefore, we chose to compare the plates against the room temperature 89h growth as the colony density was more compatible.

Compared to the room temperature α plate that grew over the weekend, where only α2 was fully pink, all of the colonies on the α plate that grew in 37℃ for 24h were more pink (figure 12). These plates suggested that temperature did have an effect on the expression of mRFP1.

From the plates, it could be suggested that 37 and 42℃ had the greatest activation of expression of mRFP1. To try and distinguish between 37 and 42, we put them in the specific incubators for another 24h.

All 6 samples on plate α seem to have the highest fluorescence on either 37℃ or 42℃ plates. For some samples e.g. α3 and 9, more cells seemed to be fluorescent on the 37℃ plate. This aligns with AIS-China 2023 iGEM team's characterisation of BBa_C0051, where fluorescence/OD600 peaks at 37℃.
On the other hand, β10 seems to have the higher fluorescence at 23℃, and fluorescence decreases as temperature increases.

We wanted to test if the increase in fluorescence was due to temperature reducing TcI inhibition of mRFP1 expression or due to the faster cell growth. To do this, the liquid samples from over the weekend were diluted and reinoculated and transferred to the same 4 different temperatures. After 24h, an end point reading of fluorescence and OD600 was taken using a plate reader. The data was blank corrected and relative fluorescence was calculated by:
fluorescence[sample]/(fluorescence[blank]*OD600)

However, there were a few negative values in the OD600 reading. A suggestion for this outcome is the droplets on the film cover from overfilled wells. We wished to repeat this experiment again, however after the colony PCR where 1p was identified to be short of around 200bp, we decided to move onto the next engineering cycles with the aim to repeat these experiments with fully working constructs.

Learn #3

There appeared to still be issues with our golden gate process, which included the promoter BBa_J23119. Different constructs that also used this part seemed to not function as intended. Following our troubleshooting of the 1p golden gate, we decided to begin the next cycle of designing and building in parallel to the testing of cycle 3.

This cycle also led us to think more about how best to do our validation experiments. For example, we saw brighter fluorescence on solid media at 37℃, but realised that we could not confirm that this was due to reduced TcI repression, as opposed to faster cell growth and/or global increases in protein expression. We conducted tests in liquid culture to account for cell number, but will need further controls in future experiments to correct for the general increase in transcription/translation at 37℃.

Design #4

Design #4 was identical to the previous cycle, except the promoter BBa_J23119 was switched to BBa_J23109 following the troubleshooting. We chose this promoter because we wanted a constitutive promoter that was characterised with moderately high expression.

Plasmid	Part	Source	Notes
TcI gene expression (1p V3)	ChlR and CloDF13 ori backbone	From instructor	PCRed up to introduce BsaI cut sites and appropriate overhangs
	Anderson promoter	Distribution kit	BBa_J23109
	RBS	Distribution kit	BBa_J435374 (BC_B0032)
	TcI gene	Synthesised	Ordered as a double-stranded gene fragment from Twist
	Double terminator	Distribution kit	BBa_J428092 (BBa_B0015)
TcI-dependent mRFP1 expression (2p V1 and V2)	KanR, pUC ori backbone	Distribution kit	BBa_J435330 (high copy (pUC) ori/KanR)
	TcI-dependent promoter	Distribution kit	BBa_R0051 (Cell_Comm_Promoters_BBa_R0051)
	RBS	Distribution kit	BBa_J435374 (BC_B0032)
	mRFP1	Distribution kit	BBa_J428036 (mRFP1)
	Double terminator	Distribution kit	BBa_J428092 (BBa_B0015)

Build #4

The golden gate for the new 1p design (1p V3) was constructed and validated by colony PCR (16). These plasmids were then transformed into BL21 cells by electroporation, along with 2pV1. Surprisingly, colony PCRs of the double transformants showed that the 1p in these cells was again missing a region (~150bp) (figure 26 left). One explanation for this might be homologous recombination between the two plasmids, although this is uncommon in E. coli. In the future, this could be avoided by not using the same terminator and ribosome binding site for multiple plasmids if they are being transformed into the same cell.

Test #4

The 6 colonies picked for colony PCR were also inoculated into liquid medium and grown overnight. The next day, 6uL of each culture was added to four different plates. Each plate also had an α9 colony, as it had seemed promising in cycle 3 and we wanted to repeat the testing, and a pTi colony as a positive control. This is because pTi constitutively expresses a red fluorescent protein, and would reflect the increases in protein expression/maturation that occur with temperature but are independent of TcI. Each of the four plates was kept at a different temperature (23C, 30C, 37C, and 42C), as before. The plate was then imaged after 30 hours.

A similar experiment was conducted in liquid media. For this, the overnight cultures were diluted 30X and then 500uL was added to four eppendorfs. Each set of tubes was incubated at a different temperature. After 30 hours, an endpoint plate reader measurement of OD600 and fluorescence was conducted. Building on what we did in cycle 3, we included BL21 as a negative control to account for autofluorescence, and pTi as a positive control to account for non-TcI-related temperature dependence. The high standard deviation between triplicates in our samples suggests that the noise far outweighs the signal, i.e., the samples are likely to not actually be fluorescing.

In addition to repeating the experiments of cycle 3, we also ran an extended plate reader assay to monitor real time changes in fluorescence intensity in response to temperature. This involved setting the plate reader to increase in temperature by 1C from 25 to 45 with every cycle, and then to stay at 45C for 17 further cycles. This experiment also included the older room temperature samples of cycle 3, as these would be at a similar growth phase to the overnight cycle 4 cultures. These results suggest that while some of the cycle 3 constructs increase in fluorescence with temperature, none do so significantly more than the positive pTi control. The cycle 4 constructs show very high variation, as they did in the results above. This is similar to the untransformed BL21 control, reinforcing the idea that this is due to there not being any fluorescence (so all of the “signal” is derived from random noise).

Design #5

Alongside cycle 3 and 4, we attempted another design using a combination of already acquired parts. In this design, the IDT construct (design 2) containing both TcI and eYFP transcriptional units were implemented alongside the 2p mRFP1 plasmid (design 3).

Build #5

2 variations of this design were built. The first build was the chemical transformation of IDT synthesised plasmid and miniprepped 2p plasmid into BL21 cells (labelled 'new'). The second was the chemical transformation of IDT plasmid straight into already transformed 2p cells from cycle 3 (labelled "old").
Both transformations resulted in at least 1 colony growing on the double antibiotic (chl and kan) plates (figure 18).

Test #5

Both plates were placed in the 42℃ incubator for 48h. The plate containing 'old' cells formed a dark pink colour in the centre (figure 31 bottom right), which was darker than the colour in cells from cycle 3.

This suggested that there was some temperature effect on mRFP1 expression on the 'old' plate. No eYFP fluorescence from the IDT plasmid could be seen due to the autofluorescence of the LB agar.

Design #6

Primers were designed to PCR up the majority of the Design#2 construct, except for the Tac promoter. This was done by creating a forward primer immediately downstream of the promoter, and a reverse primer immediately upstream. Both primers introduced recognition sites for BbsI (the codon optimisation done to reduce complexity previously had introduced BsaI sites, so these could not be used for assembly). The overhangs were designed such that the 3' end of the digested amplicon would anneal to the 5' end of the BsaI-digested promoter from the kit part, and the 5' end would anneal to the 3' end of another oligo. This oligo was designed to act as a spacer, taking 100bp of sequence from the AConR-end part in the Open Yeast collection. As mentioned, its 3' end anneals to the digested amplicon, while it's 5' end would anneal to the 3' end of the promoter. Standard overhangs used in the kit mean that this method could be used for any of the promoters included in the distribution kit, but we planned to use the constitutive Anderson promoters.

A similar set of primers were designed to allow us to swap the coding sequence of the construct using restriction-ligation. However, here we were looking to switch out the coding sequence change from eYFP to something that is easier to detect against LB-agar autofluorescence, e.g., RFP. It could also be to introduce a fluorescent-protein quencher system, which would work with EPG Cycle 4 to create an AND gate for 2D spatial targeting.

Build #6

This plasmid would have been assembled via a restriction ligation (not a Golden Gate). The plasmid from the distribution kit containing the promoter would be digested with BsaI, while the IDT constructs would have been amplified using the designed primers and then digested with BbsI. Both of these digestions would be run on a gel and the desired fragment would be gel purified. Then, the digested IDT amplicon would undergo a dephosphorylation reaction to avoid self-annealing in the ligation step. Meanwhile, the spacer, which was ordered as two single stranded oligos (so that no digestion is needed to create the overhangs), would be put together in an oligo annealing reaction. Finally, the dephosphorylated IDT construct would be mixed with the spacer and promoter in a ligation reaction, which would then be transformed into DH5a cells to plate on an LB+ampicillin plate.

In order to change the coding sequence, a similar process would be used. The amplicon would be BbsI digested, gel purified, and dephosphorylated. Meanwhile, the ribosome binding site and coding sequence would be purified from the distribution kit and digested with BsaI. All three digestions could then be mixed together in a ligation reaction, then transformed and purified from cells.

Through our cycles, we aimed to construct a variety of TcI constructs that could ultimately be activated through magnetic fields heating up magnetic nanoparticles. We were successful in the transformation of cycles #2-5, as the cells grew on antibiotic plates. We also developed our protocols for testing thermal activation in both solid and liquid mediums. As we came across many roadblocks in areas such as transformations, miniprep, and Golden Gates, we carried out cycles in parallel in the aim of characterising the TcI plasmid using our hardware design. Following a future cycle, our constructs would be tested with the incubator protocol used in cycles #3-5, followed by incubation on a magnet.

Superparamagnetic nanoparticles are magnetized in the presence of magnetic fields, and thus a slowly alternating magnetic field results in the oscillation of the particles. If bound to proteins on the cell surface, these oscillations can pull/push on the proteins to alter the tension on the cell wall/membrane.

Wsc1 is a yeast cell membrane protein that acts as a sensor for the cell wall integrity pathway by activating the Rom2 GEF and thus the downstream MAPK signaling cascade, regulating the expression of at least 25 genes implicated in cell wall biogenesis (Philip and Levin, 2000). An elongated form of Wsc1 was engineered by Dupres et al (2009), such that it extended out of the cell wall with a His tag that could interact with their NTA-Ni⁺ functionalized atomic force microscopy tips.

Design #1:

Our initial plan was to repeat what was done by Dupres et al to generate a plasmid expressing the elongated, His-tagged, Wsc1. This involved the PCR amplification of native genes from the genome and the manipulation of yeast homologous recombination to create fusion proteins. The protocol used in the paper is described below:

1. PCR amplify Wsc1 from genome with oligonucleotides 06.37/06.38.
2. PCR product digested with BamHI and SalI.
3. Cloned into respective sites of YCplac111 (to form pSK27).
4. Integrate URA3 marker between signal peptide and cysteine domain of Wsc1.
5. URA3 amplified from plasmid YDp-U with oligonucleotides 06.163/06.164.
6. Product from step 5 is co-transformed with pSK27 (step 3) into yeast strain HAS17-3C.
7. Transformants selected on minimal medium without leu or ura.
8. Resulting plasmid pSK30 isolated and amplified in E. coli.
9. Substitute URA3 for serine/threonine rich region of Mid2:
• Serine/threonine rich region of Mid2 amplified from plasmid pAS29 using the oligonucleotides 08.38/08.39.
• PCR product co-transformed with pSK30 (step 8) into yeast strain HD56-5A.
10. Plated on medium lacking leu supplemented with 1mg/ml 5-FOA [5-fluoroorotic acid].
11. Plasmid pBH1 (=YCpWSC1-S/T-His) recovered, confirmed by restriction analysis and sequencing to show it contains Wsc1 elongation.

We also planned to construct a reporter plasmid, using the Rlm1 promoter upstream of mRFP (Rlm1 is an autoregulator so should bind its own promoter). This would be almost entirely constructed from the Open Yeast Collection parts found in the kit, except for the Rlm1 promoter itself, which would have been amplified from the yeast genome.

Design #2:

While designing the oligonucleotides to allow us to build Design #1, we also reached out to Prof. Heinisch to ask if he could send us some of the pBH1 plasmid for elongated Wsc1 expression. He replied and offered to send us various strains and plasmids, as described in the table below. We preferred these to our designs because:

1. The strain had one of the relevant plasmids integrated into its genome, which is much more stable than trying to express two plasmids within a cell.
2. The plasmids he was sending had CEN/ARS sequence, which is also more stable and more consistent than the 2 micron origin available in the Open Yeast collection of the distribution kit.
3. There are fewer steps if we don't have to construct the plasmid ourselves, saving time and making troubleshooting easier if the construct doesn't work.

Build #2:

While transforming pBH1 into HAS100L would have had the same effect as transforming pHPS100H into Y1, we chose to do the latter due to marker compatibility. We called the Y1 strain transformed with pHPS100H Y100H. Transformations were plated on a triple drop-out plate, and colonies grew, indicating that the transformation was a success.

Test #2:

Initial testing involved verifying if the genetic pathway worked, independent of magnetic nanoparticles and/or fields. This involved subjecting the cells to various conditions under which the cell wall integrity (CWI) pathway is known to be activated. One of these conditions is incubation at 37°C (as opposed to the 30°C that is standard for S. cerevisiae). HAS100L was used as a positive control here, since it contains the same reporter as in Y100H except integrated into the genome rather than as a plasmid. HAS100L also lacks the elongated Wsc1, but this should not affect its response to non-magnetic activation of the CWI pathway. Y1 has elongated Wsc1 but lacks the lacZ reporter. It is used as a negative control in the test to verify that untransformed Y1 will not respond to the X-gal test, which is a secondary indication of the successful transformation with the plasmid pHPS100H.

The result showed that after overnight incubation at 37°C, HAS100L and Y100H colonies turned blue after being treated with X-gal, while Y1 colonies remained white.

This experiment confirmed that our transformation was successful and that our reporter system successfully expressed lacZ when an osmotic pressure change was applied to the cells.

Learn #2:

Our experiment demonstrated that the transformation of Y1 with the reporter plasmid pHPS100H was successful, and the new strain could respond to the osmotic pressure change around cells.

The next step is to investigate other conditions that could lead to the activation of the CWI pathway and summarize them before conducting the magnetic activation. This is to avoid obtaining false positives and drawing incorrect conclusions in the final activation with magnetic fields and magnetic nanoparticles.

Design #3:

In one of our repeated experiments to test CWI pathway activation at 37°C with X-gal, we incubated one masterplate at 37°C and another masterplate at 30°C as a negative control. The incubation time was extended to over the weekend (i.e., over 3 nights).

After treating colonies with X-gal, we noticed some unusual results in two masterplates.

The results suggested that for long-period activation, cells incubated at 30°C may have higher beta-galactosidase activity compared to cells incubated at 37°C. This is not what we expected and suggests that CWI pathway activation might be affected by incubation time and temperature. We need to set up new experiments to further investigate the conditions for the activation of the CWI pathway in S. cerevisiae.

Build #3:

To further investigate the effect of temperature and incubation time on beta-galactosidase activation, four masterplates were set up: 2 incubated at 30°C, and 2 incubated at 37°C. After one night, one plate was taken from both the 30°C and 37°C groups and treated with X-gal. The remaining plates were taken out and treated with X-gal after two nights.

Test #3:

After two nights, we obtained results from the planned experiment.

The results (Figure 35) showed that for overnight incubation, 37°C had more activation than 30°C, whereas two nights of incubation resulted in 30°C having more activation than 37°C. This may be explained by the fact that growth also activates the CWI pathway, and so, over a longer incubation period, better growth at 30°C outweighs the effect of temperature. It also emphasized that there is a noticeable level of background signal under standard conditions, highlighting the importance of controls in later experiments.

Observing signals in Fig. X, we noted that for overnight incubation, Y100H had higher beta-galactosidase activity than HAS100L. For 2-nights incubation, HAS100L showed higher beta-galactosidase activity. We decided to conduct another experiment to investigate if the lacZ activity differs between different strains.

The experiment was conducted by adding 5 μl of X-gal stock solution to colonies after they formed. Plates were incubated at 30°C for 8 days to ensure they hydrolyzed all X-gal provided.

Under long-term incubation (36), HAS100L exhibited much higher beta-galactosidase activity. This was reassuring because, for HAS100L, the lexA-Rlm1-lacZ reporter is integrated into its genome, while for Y100H, the reporter is expressed as a low-copy plasmid. If the results had been the other way around, we might have needed to transfer the pBH1 coding sequence into a backbone compatible with HAS100L, rather than continuing with Y100H.

We also tested the response of the genetic pathway to mechanical stress in liquid medium using two different methods: vortexing (mechanical stress) and water bath (osmotic pressure change).

Colonies were taken from a fresh Y100H plate and re-suspended in 750 μl of triple amino acid drop-out liquid medium. Three tubes were set up: water bath at 39°C for 20 mins, vortexing for 10 mins, and a negative control incubated at room temperature. All tubes (except the negative control) were then placed in a 37°C shaker for 24-hour incubation.

The results suggest that X-gal testing works for a simple liquid-based method but takes a long time if no additional treatment is employed to change the cell membrane permeability of S. cerevisiae cells.

Learn #3:

We conducted several experiments to investigate different conditions that may affect CWI pathway activation and beta-galactosidase activity. Having validated our genetic pathway, we were ready to test the magnetic activation of our pathway.

Design #4:

Before conducting the magnetic activation test on our engineered S. cerevisiae cells, we needed to investigate the behavior and toxicity of the 250 nm Ni-NTA coated magnetic nanoparticles (MNPs) we would use.

First, we aimed to view them under an appropriate microscope to confirm attachment to S. cerevisiae cells. Additionally, we planned to conduct a co-incubation test between MNPs and yeast cells to investigate toxicity and ensure that cells would not die after MNP treatment.

After validating MNPs, we needed to set up an appropriate test to attach MNPs to His-tagged Wsc1 on cells and apply an external magnetic field to activate the CWI pathway.

Build #4:

250 nm NTA-Ni coated MNPs (nanomag-D, Micromod Partikeltechnologie GmbH) were used. These MNPs are water-soluble and could be viewed directly under a standard light microscope. For visualization, 2 μl of MNPs were taken and viewed under a light microscope with a 100x objective lens.

To bind MNPs with His-tagged Wsc1 on yeast cells, 10 μl of MNPs (10 mg/ml) was mixed with 2 ml of liquid culture of Y1 and allowed to incubate for 2 hours for complete binding. 5 μl of the mixture was taken and viewed under the light microscope with a 100x objective lens.

Video 1 & 2: MNPs bound to cells doing Brownian motion on the surface of cells.

In video MNPs_1, MNPs are seen as small, fast-vibrating particles bound to cells, performing Brownian motion on/around cells. In video MNPs_2, MNPs are distinguished from yeast vacuoles; bigger, darker, stationary/slow-moving spots are vacuoles, while small, fast-moving spots are MNPs.

A toxicity test was set up based on OD600 value analysis. Overnight cultures of Y1, Y100H, and HAS100L (OD600 ~1.5) were diluted 30x. They were then added to a 96-well clear plate (Greiner) with the relevant media and MNPs, according to the layout below:

The plate reader was set to incubate at 30°C, measuring absorbance at 600 nm every 10 minutes for 289 cycles (48-hour runtime) with double orbital shaking at 200 rpm between cycles.

A smaller masterplate was built for the solid-based method of magnetic activation: a 35 mm petri dish divided into two separate areas. For each area, 6 μl of HAS100L/Y100H liquid culture was added as a single droplet, incubated at room temperature for ~15 mins to settle, then incubated at 30°C overnight to form colonies.

Test #4:

OD600 values were imported as raw data to show the growth of different S. cerevisiae strains over ~48 hours.

Y100H was incubated with different concentrations of 250 nm MNPs over 48 hours. OD600 values were imported and plotted as the toxicity curve.

In magnetic activation experiments, HAS100L acted as a negative control because it contained the same reporter system for the CWI pathway but should not experience magnetic field-dependent CWI activation. MNPs and X-gal were added to colonies on two 35 mm plates, one colony each of Y100H and HAS100L. One plate was kept at room temperature, while the other was subjected to a low-frequency, high-strength magnetic field (100 mT, 1 Hz). Immediately after an hour of activation, a small amount of blue was visible around the edge of the activated colonies. Both plates were incubated at 30°C, and long-term incubation led to a more pronounced difference between the activated and non-activated plates.

Learn #4:

The growth curve results showed that Y100H had a higher growth rate than HAS100L, supporting the result obtained in Test 3, where Y100H exhibited higher beta-galactosidase activity after overnight incubation. Y100H had a slower growth rate than Y1, likely due to the expression of an additional plasmid (pHPS100H) in Y100H compared to Y1. Toxicity results indicated that 250 nm Ni-coated MNPs had minimal effects on the growth of the yeast strains within the concentration range tested.

Figure 43 shows a qualitative increase in beta-galactosidase when colonies are placed under a magnetic field, confirming our hypothesis. Interestingly, HAS100L experienced similar activation levels, despite not having a His-tagged Wsc1 that directly interacts with the Ni-NTA nanoparticles. This may be due to nanoparticles binding to native histidine-containing cell surface proteins and exerting force on the cell wall through them, or it may be that the movement of nanoparticles in the media causes mechanical stress without specific interactions with the cell.

Our experiments have been qualitative, and more quantitative tests are needed given the subjectivity in comparing the 'blueness' of samples. The optimal approach would be in liquid culture, allowing normalization against cell number. Additionally, it would be beneficial to correct for the heating effect caused by the magnetic field, which is crucial for integrating the pathway into an AND gate with thermal activation. Adding sorbitol to the medium could balance the osmotic pressure and reduce temperature sensitivity, as suggested by Figure 44 (Straede et al., 2007).

Design #1

We required an electronics design to generate a high frequency magnetic field in our chosen region. We designed this based on a zero voltage switching circuit used for induction heaters. This circuit is designed to cause an inductor and capacitor to resonate at their resonant frequency (see hardware page). Our primary design challenge to achieve this was to develop an inductor/capacitor circuit that could resonate at an appropriate frequency/power to generate our magnetic field. We used equation models and finite element analysis to generate these designs. The tradeoff to balance was that by increasing the number of turns in the electromagnet, we also reduced the oscillation frequency. To generate effective heating both the power and frequency needed to be high.

Build #1

We built 7 iterations of our coil design outlined below:

Test #1

Each of these designs were tested using simple heating protocols on our magnetic nanoparticles where we placed 0.2ml of each MNP solution into an eppendorf and powered the electromagnet, measuring heat generated.

Learn #1

We were able to demonstrate that heating was possible and that out SAR values were comparable to the literature using significantly lower cost hardware. Further development would be needed to increase scale/reduce MNP concentration but the power requirements meant they were out of reach for this project.

Design #2

In parallel, we also needed to develop an electronics design to generate static and low frequency oscillating magnetic fields. Our design to achieve this used low cost H-Bridge motor drivers which were capable of driving and switching the current we needed. They are also effective because they are designed to drive the inductive loads of motors.

Build #2

We then build this design:

For more information on this build, see our hardware page.

Test #2

We tested our design by applying a 1Hz oscillating magnetic field to our Wsc1 construct and measuring the response of the pathway.

The activation was sustained for one hour. For further details on the process and results, please refer to the Wsc1 DBTL cycles.

Learn #2

We were successfully able to trigger downstream pathway activation via magnetic nanoparticle force transduction so we were confident we could move onto focussing on the spatial activation hardware.

Design #3

We wanted to develop an automated way to apply our targeted magnetic fields for precision, repeatability and reproducibility so we needed to design a device to achieve this. In the design process we made several important decisions about how this device would work including the mechanisms necessary and the sensor choice (read more on our hardware page.)

Build #3

After developing a complete CAD model of the design, we sourced and made the parts and then assembled them together:

Test #3

To test the design, we tested the operation of each of the components and confirmed we could achieve the measurement we needed. We also completed some user testing with the device with 3 different test groups.

Learn #3

Some of the design decisions we made in this initial phase were shown to be effective but a number required improvement. In particular, we needed to improve the mounting of electromagnets and lighting/imaging arrangement for measurement. From the user testing, we decided to develop a graphical interface for ease of use and integrate the electronics into the box to improve reliability.

Design #4

Building on what we learned from the first iteration of the device, we set about developing a second design. We started by improving the motor mount design to reduce the flex when moving by adding more points of contact with the stage. We also moved the blue fluorescent illumination source to the top so we could darken the base of the box completely for imaging purposes. We also designed a new box section with custom shelving for our electronics.

Build #4

We then built this design and documented the process for our hardware guide:

Test #4

We managed to operate all of the components of our device and run some initial calibration and activation tests.

Learn #4

In the future, we’d have liked to run more integrated testing between our hardware and our constructs and to characterise the resultant spatial precision possible. This would have enabled us to better inform the downstream applications of this device.

MagentaBOX and the designs we developed for each electromagnet are cost effective and easy to use set of tools for continuing magnetogenetics research. The DBTL cycles we have acheived have significantly aided in increasing the quality and usability of these hardware tools. There remain a number of components that we would have liked to develop further - primarily in characterising and improving spatial precision of our designs both in 1 and multiple dimensions. We hope that future teams and researchers will continue this work forwards.

EPG:
Krishnan, V., Park, S.A., Shin, S.S. et al. Wireless control of cellular function by activation of a novel protein responsive to electromagnetic fields. Sci Rep 8, 8764 (2018). https://doi.org/10.1038/s41598-018-27087-9

HSF:
Santoro, N., Johansson, N., & Thiele, D. J. (1998). Heat Shock Element Architecture Is an Important Determinant in the Temperature and Transactivation Domain Requirements for Heat Shock Transcription Factor. Molecular and Cellular Biology, 18(11), 6340–6352. https://doi.org/10.1128/MCB.18.11.6340

TlpA and TcI:
Hurme, R., Berndt, K.D., Normark, S.J., Rhen, M., 1997. A Proteinaceous Gene Regulatory Thermometer in Salmonella. Cell 90, 55–64. https://doi.org/10.1016/S0092-8674(00)80313-X

Piraner, D., Abedi, M., Moser, B. et al. Tunable thermal bioswitches for in vivo control of microbial therapeutics. Nat Chem Biol 13, 75–80 (2017). https://doi.org/10.1038/nchembio.2233

Piraner, D.I., Wu, Y., Shapiro, M.G., 2019. Modular Thermal Control of Protein Dimerization. ACS Synth. Biol. 8, 2256–2262. https://doi.org/10.1021/acssynbio.9b00275

Valdez-Cruz, N.A., Caspeta, L., Pérez, N.O. et al. Production of recombinant proteins in E. coli by the heat inducible expression system based on the phage lambda pL and/or pR promoters. Microb Cell Fact 9, 18 (2010). https://doi.org/10.1186/1475-2859-9-18

Wsc1:
Philip, B. and Levin, D. E. (2001) ‘Wsc1 and Mid2 Are Cell Surface Sensors for Cell Wall Integrity Signaling That Act through Rom2, a Guanine Nucleotide Exchange Factor for Rho1’, Molecular and Cellular Biology, 21(1), pp. 271–280. doi: 10.1128/MCB.21.1.271-280.2001.

Dupres, V., Alsteens, D., Wilk, S. et al. The yeast Wsc1 cell surface sensor behaves like a nanospring in vivo. Nat Chem Biol 5, 857–862 (2009). https://doi.org/10.1038/nchembio.220

Straede, A., Corran, A., Bundy, J., Heinisch, J.J., 2007. The effect of tea tree oil and antifungal agents on a reporter for yeast cell integrity signalling. Yeast 24, 321–334. https://doi.org/10.1002/yea.1478

Burn, Sally F. "Detection of β-galactosidase activity: X-gal staining." Kidney Development: Methods and Protocols (2012): 241-250.

Kippert, Fred. "A rapid permeabilization procedure for accurate quantitative determination of β-galactosidase activity in yeast cells." FEMS microbiology letters 128.2 (1995): 201-206.

Rotherham, Michael, et al. "Remote regulation of magnetic particles targeted Wnt signalling for bone tissue engineering." Nanomedicine: Nanotechnology, Biology and Medicine 14.1 (2018): 173-184.

Watanabe, Yasuyuki, et al. "Characterization of a serum response factor-like protein in Saccharomyces cerevisiae, Rlm1, which has transcriptional activity regulated by the Mpk1 (Slt2) mitogen-activated protein kinase pathway." Molecular and cellular biology 17.5 (1997): 2615-2623.

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