iGEM 2024 — Team cambridge

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## Weekly Progress

Click on each week for more details.

Before Lab work (Dec 23-July 24)

##### Recruitment (December-January)

* Applications to join the team were due by 1st December
* Interviews with members of the CU Synthetic Biology Society committee were carried out during December
* The team was finalised in mid-January

##### Brainstorming and training (January-March)

* Weekly brainstorming sessions to discuss and develop project ideas
* Information sessions on synthetic biology, iGEM, human practices, etc.,
* Meetings with academics and industry experts for advice on idea feasibility

##### Detailed project design (Early July)

* Literature review on previous methods in magnetogenetics and possible pathways that could be manipulated e.g. EGFR, EPG, Hog1, HSF1, MscS, Piezo, Ste2, TcI, TlpA, Wsc1
* Literature review on magnetic nanoparticles and their purchase and synthesis
* Meetings with academics for advice on project feasibility and suggestions

##### Getting ready for lab work

* Risk assessment for iGEM and the Department of Engineering lab space
* Created template for wet lab notebook
* Discussed logistics of ordering reagents and plasticware with advisors
* Parts mining for plasmid design through iGEM registry and scientific papers
* Workshop on primer design
* Induction and training in lab
* Researching and compiling protocols
* Inventory of items left by previous iGEM teams
* Design and ordering of primers and gene fragments for the construction of plasmids needed to test EPG, TlpA, and TcI
* Design of AFM circuit and fluorometer

##### Outreach

* Designing materials for Smallpeice outreach event

Week 1 (15/07-21/07)

##### Wet lab

* Receiving, suspension, and storage of primers
* Receiving and preservation of required E. coli and S. cerevisiae strains for TlpA/TcI, Wsc1, HSF, and EPG pathways
* Test of antibiotics that are required for transformation
* Preparation of competent cells for Golden gate
* PCR of parts required for Golden gate

##### Hardware

* Box no.1, for imaging plates on the large electromagnet, constructed
* Raspberry Pi 3 obtained

##### Modelling

* Modelling workshop with instructor
* Construction of ODE model for TlpA, TcI, and Wsc1 started
* Literature review on model parameters

##### Human Practices

* Start reading on neuroscience, organoid, and policies
* Start communication with academics

Week 2 (22/07-28/07)

##### Wet lab

* Antibiotics testing
* Test transformation of pUC19 and luciferase from the kit to test the competency of competent cells and efficiency of chemical transformation protocol
* Start transformation of kit parts required for all pathways and get ready for the golden gate
* Yeast HSF pathway construction starts
* 250 nm magnetic nanoparticles visualisation with a light microscope

##### Hardware

* ZVS heating of ferrofluids obtained from Cavendish lab
* ZFS coil parameter analysis
* Design of box with moving magnets and imaging system

##### Modelling

* Construction of model calculating forces generated by 250 nm magnetic nanoparticles started

##### Human Practices

* Detailed feedback from researchers in organoid development and neuroscience
* Plan for structures of human practices deliverable

Week 3 (29/07-04/08)

##### Wetlab

* Attempting minipreps on previously transformed cells
* Validation of miniprep parts on gel
* Troubleshooting chemical transformation
* Testing and trials of electroporation
* Continuous working on TlpA/TcI pathway construction
* More MNP observation under light microscope, observing random movement change to biassed movement upon application of a magnetic field

##### Hardware

* Coil design iterations

##### Modelling

* Modelling of magnetic field shape and strength, used to inform coil design iterations
* Modelling of nanoparticle heating and dissipation of heat across plate
* Modelling of the HSF and Wsc1 pathways

##### Human Practices/Outreach

* Making the table of stakeholder areas and people we have communicated with
Making a comparison table of sonogenetics, optogenetics, magnetogenetics, and chemical methods * Contacting more stakeholders in a more diverse range
* Biomedical engineering summer course outreach event

Week 4 (05/08-11/08)

##### Wet lab

* Chemical transformation has relatively low efficiency. Electroporation works better. We’re switching the transformation method to electroporation
* Previous miniprep is not working and we need to re-transform all parts required.
* Construction of EPG pathway starts
* First attempt at golden gate assembly parts available for TlpA/TcI

##### Hardware

* Testing of relays, servo, camera, thermal camera, motors, and display
* Initial development of GUI for control and measurement of magnet

##### Modelling

* Further iterations on modelling of TlpA/TcI, HSF, and Wsc1 pathways
* Calculation of magnetic field gradient
* Calculation of force acting on magnetic nanoparticles in water
* Modelling of further EPG constructs, with LacI and TEV

##### Human Practices

* Meeting with Stuart Ibsen about sonogenetics - costs of technology, how the technology works, use cases.
* Meeting with Mightex about optogenetics - found out rough costs of equipment and cost ranges. Found out more about who is using optogenetics (mostly universities).

Week 5 (12/08-18/08)

##### Wet lab

* Golden gate of EPG started
* MNPs attachment test to see if MNPs bind to his-tagged Wsc1
* Preparation and testing for yeast Leu- Ura- His- selection plate
* Transformation of Y1 for Wsc1 pathway
* Miniprep is not working, figuring out what's the problem and changing the kit for miniprep
* Troubleshooting of previous TlpA / TcI golden gate by gel and digest validation

##### Hardware

* Development of imaging system
* Further design of the “magenta” box with the moving magnet

##### Modelling

* Further modelling on HSF and discussion with the instructor about the temperature function
* EPG-TEV model constructed

##### Human Practices

* Meeting set up with neuro-oncologist at Addenbrooke’s next Tuesday 10am
* Responses from CAR-T cells researcher
* Girls in Math educational event at St Johns’ college Cambridge

Week 6 (19/08-25/08)

##### Wet lab

* Transformation of Y1 with pHPS100H for wsc1 construct
* Testing of X-gal and sorbitol on HAS100L
* Golden gated plasmids are not working. We need to check from the parts, miniprep, transformation, golden gate with gel validation to see what went wrong

##### Hardware

* Construction of the “magenta” box with the moving magnet and imaging system

##### Modelling

* Initial model of HSF finished and had a discussion with the instructor
* Integrating HSF model with thermal magnetic modelling

##### Human Practices

* Meeting with Dr Rajesh Jena (neuro-oncologist) who had a lot of interesting thoughts about glioblastoma recurrence prevention and CAR T-cell therapy application ideas
* Draft of Education page

Week 7 (26/08-01/09)

##### Wet lab

* Successful transformation of Y1 with pHPS100H. Conducting sorbitol and X-gal test on Y100H to validate the construct is working
* MNPs binding test to Wsc1
* Golden gate troubleshooting
* Toxicity test of 20 nm MNPs on E. coli DH5alpha and BL21
* Re-transforming, miniprep, digest, and golden gate the parts

##### Hardware

* Testing on a large coil to validate if a magnetic field with a low frequency, high field strength could be produced
* 20 nm MNPs heated up when a magnetic field with high frequency and high field strength was applied

##### Modelling

* Improvement of TcI and Wsc1 modelling

##### Human Practices

* Preparation of posters and slides that will be used for UK 2024 iGEM meetup at Sheffield

Week 8 (02/09-08/09)

##### Wet lab

* The transformation and direct miniprep did not work well, and we decided to colony PCR the parts we need and validate it
* Figuring out a working protocol to conduct X-gal testing on yeast
* Testing of sonicator on liquid X-gal testing with yeast
* Wsc1 MNPs testing with magnetic field in a liquid based culture
* Toxicity testing of yeast with 250 nm MNPs
* Continuous attempts of golden gate of EPG parts

##### Hardware

* Set up the algorithm for the camera fluorescence measuring
* Movement of magnets, and calibration with limit switches
* Testing on the motor.

##### Modelling

* Further improvement of modelling of TlpA/TcI and EPG

##### Human Practices

* Writeup in progress
* UK iGEM Meetup 2024 this weekend

Week 9 (09/09-15/09)

##### Wet lab

* Colony PCR of EPG, validation with gel, transformation with BL21 and test with nanoluc
* MNPs-resuspended in LB agar and testing the bacterial growth and heating of the plate
* Arrival of synthesised TlpA and TcI constructs
* Miniprep of TlpA and TcI constructs from Dh5alpha
* Wsc1 MNPs testing with magnetic field in both solid and liquid method

##### Hardware

* Software set-up and testing interactions of the “magenta” box
* Camera test and calibration

##### Modelling

* Summarising TlpA and TcI modelling

##### Human Practices

* Writeup approaching the end

Week 10 (16/09-22/09)

##### Wet lab

* Colony PCR, miniprep, and gel validation of EPG and TcI construct
* Troubleshooting of the TcI construct
* Heat-testing of TcI and testing of EPG luminescence with magnet

##### Hardware

* New coil design and ferrofluid heating test
* Testing of three constructed boxes with instructors and getting user’s feedback

##### Modelling

* Summary of all models (TlpA/TcI, wsc1, HSF, magnetic force, magnetic heating) and final writeup planned

##### Human Practices

* Write-Up content done. Planning the final format to be displayed on wiki

Week 11 (23/09-29/09)

##### Wet lab

* Colony PCR, miniprep, and gel validation of TcI construct
* Repeats of TcI heat-activation to test new golden gate 1pV3
* Miniprep of some constructs for sequencing
* Summarising all testing results and uploading new basic parts/constructs to iGEM registry

##### Hardware

* Construction of the final box finished
* Hardware writeup in progress

##### Modelling

* Summarising all models. The final write up is uploaded to wiki

##### Human Practices

* Write-Up content uploaded to wiki

## Detailed wet lab notebook

Click on the contents link to be taken directly to the page or scroll down for the second part of the notebook.

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## EPG

### Introduction

EPG is a novel magnetosensitive gene discovered around 2018 (Krishnan et al., 2018),
with demonstrated potential for controlling cellular activities under a magnetic field. However, the mechanisms
underlying EPG's mode of action remain largely unexplored. As a result, new designs are required to effectively
integrate EPG into our system.

### Cycle 1: EPG - NLuc construct TE CE LE Cycle 1

#### 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.

![EPG-Nanoluc expression plasmid](https://static.igem.wiki/teams/5154/engineering/t7te.png)
![Positive control plasmid](https://static.igem.wiki/teams/5154/engineering/tr.png)
![Negative control plasmid](https://static.igem.wiki/teams/5154/engineering/tev-negative-control.png)

Our EPG-Nanoluc expression plasmid, positive control plasmid and negative control plasmids.

#### 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.

![failed gel showing the digest is not working](https://static.igem.wiki/teams/5154/engineering/failed-gel.jpg)
![not working miniprep](https://static.igem.wiki/teams/5154/engineering/miniprep.png)

Gel image showing the miniprep of the parts retrieved from distribution kit is not working

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.

### Cycle 2: EPG - NLuc construct TE CE LE Cycle 2

#### 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).

![Colony pcr for the goldengate related to EPG nanoluc](https://static.igem.wiki/teams/5154/engineering/nanoluc-colony-pcr.png)

Colony PCR of the EPG-Nanoluc constructs, showing we are having inserts with a correct length

![Cloning plasmid sequencing](https://static.igem.wiki/teams/5154/engineering/ce-sequencing.png)
![Expression plasmid sequencing](https://static.igem.wiki/teams/5154/engineering/te-alignment.png)
![Another version of expression plasmid](https://static.igem.wiki/teams/5154/engineering/le-alignment.png)

Sequencing result of our Goldengate constructs. TE has a single mutation in the promoter region, while LE have promoter and RBS region missing.

#### 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.

![first test carried out with round magnets](https://static.igem.wiki/teams/5154/engineering/simple-activation.jpg)

Initial Magnet activation test of EPG. Two round activation have been applied

#### 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.

### Cycle 3: Finding feasible protein fusion

#### 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](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%7D%7D#Magneticepg ) for more information

![Alignment of alphafold result of EPG-LacI](https://static.igem.wiki/teams/5154/engineering/alphafold.jpg)

Alignment of the Alphafold3 predicted structure of EPG-LacI fusion protein. The spatial hinderance and strain in EPG arms cause us to give up this approach.

#### 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.

### Cycle 4: EPG - TEV construct GG01-05

#### 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.

![F5-TEV-EPG construct](https://static.igem.wiki/teams/5154/engineering/f5-epg.png)
![TEV Positive control construct](https://static.igem.wiki/teams/5154/engineering/tev-positive-control.png)
![TEV Negative control construct](https://static.igem.wiki/teams/5154/engineering/tev-negative-control.png)
![R5-TEV-EPG construct](https://static.igem.wiki/teams/5154/engineering/r5-epg.png)
![GFP-REACh2 TEV cut reporter construct](https://static.igem.wiki/teams/5154/engineering/gfp-reach2.png)

Construct designed and needed for characterizing EPG-TEV fusion protein.

#### 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.

![EPG-TEV construct colony pcr](https://static.igem.wiki/teams/5154/engineering/tev-colony-pcr.png)

Gel image of the colony PCR of EPG-TEV constructs. No band have been seen and we tried this multiple times. It seemes like we have broken enzyme or other issues

#### 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.

### Cycle 5: Testing EPG-Nluc construct TE LE with different hardware

#### 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

![Non-Adjusted activation signal](https://static.igem.wiki/teams/5154/engineering/non-adjusted-mid-activation.png)
![Adjusted activation signal](https://static.igem.wiki/teams/5154/engineering/adjusted-mid-activation.png)

Activation result of EPG-Nanoluc under different strength of magnetic field. Top: Unadjusted result. Bottom: Adjusted result. Peak strength is all 100

![initial activation signal](https://static.igem.wiki/teams/5154/engineering/initial-activation.jpg)
![double activation signal](https://static.igem.wiki/teams/5154/engineering/double-activatoin.jpg)

Additional testing done with EPG-Nanoluc. Top: initial activation directly apply before the plate go into plate reader. Bottom: Double activation of the plate.

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](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%7D%7D#Magneticepg) for more information.

### Conclusion

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.

## Thermal - *S. cerevisiae*

### Introduction

The rapid vibration of small (10-20nm) magnetic nanoparticles (MNPs) under a magnetic field causes heat
dissipation. Many naturally occurring proteins take advantage of the conformation change caused by temperature
shifts to act in a temperature-dependent manner. In particular, increases in temperature can disrupt
multimerisation, which has a significant impact on transcription, as transcription factors often bind DNA as
oligomers.

The native yeast HSF actively upregulates its targets under heat shock by binding a cis-regulatory sequence in
their promoters (Santoro, Johansson and Thiele, 1998). Other thermosensitive transcription factors act in the
opposite way to repress non-essential protein production under heat stress.

### Cycle 1

#### 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.

## Thermal - *E. coli*

### Introduction

TlpA and TcI are both repressor proteins which bind as dimers to the DNA at room temperature. Under heat shock,
these repressors uncoil and unbind, allowing expression of the downstream coding sequence e.g. a fluorescence
protein.
TlpA (from Salmonella typhimurium) binds as a dimer to repress its targets at room temperatures, but uncoils and
unbinds under heat shock at 42C (Hurme et al, 1997; Piraner et al, 2016; Piraner, Wu and Shapiro, 2019). Similarly,
TcI is a thermolabile mutant form of the cI protein of bacteriophage lambda, which unbinds its operon at
temperatures above 37C (Valdez-Cruz et al, 2010).
Through our DBTL cycles, we aimed to demonstrate the thermal activation of the fluorescent output.

For more details on the background of these proteins, please refer to the [design](https://2024.igem.wiki/cambridge/design#thermal) page.

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 certain parts from the kit (figure
22) - particularly the Tac promoter and the T7 terminator for E. coli, and various parts in the open yeast
collection.

### Cycle 1: Two plasmid system with inducible promoter

#### Design #1

![SBOL diagram of 2 plasmids, one coding for TlpA, another for eYFP](https://static.igem.wiki/teams/5154/sbol-design1.png)

Biorender SBOL design of cycle 1 plasmid design

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

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 certain parts from the kit
(figure 12) - particularly the Tac promoter and the T7 terminator for E. coli, and various parts in the open
yeast collection.

![Gel electrophoresis, with wells labelled according to well index of part from the distribution kit and colour coded based on whether or not gel indicates successful extraction ](https://static.igem.wiki/teams/5154/kitpart-gels.png)

Kit parts were transformed into DH5alpha, miniprepped, and digested with PaqCI. The digestion
product was run on an agarose gel at 90V for 40 minutes. Figure 12 shows the results, with the wells labelled
according to the index of the sample in the distribution kit. Lane1:Hyperladder 1kb; Lanes 2 and 3: E18
(OYC-CamR); Lane4: G24 (pWV01-ori); Lane5: Blank; Lanes 6 and 7: M6 (Sc-pTDH3); Lane8: O13 (BN1\_BCD2\_His);
Lanes 9 and 10: O24 (oriT); Lane 11: B1 (Sc-KanRmarker); Lane 12: C19 (Tac promoter); Lane 13: D1 (Sc2micron);
Lane 14: O15 (T7 LacO promoter); Lane 15: Blank; Lane 16: Hyperladder 1kb

#### 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.

![Graph of DNA concentration against OD600](https://static.igem.wiki/teams/5154/engineering/cycle3/test1-dna-concentration-ng-l-against-od600.png)

![Graph to show DNA concentration and absorbance ratios for different miniprep kits.](https://static.igem.wiki/teams/5154/engineering/cycle3/test1-plasmid-nanodrop-data-for-each-miniprep-kit.png)

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.

### Cycle 2: One plasmid system with inducible promoter

#### Design and Build #2

![SBOL diagram of plasmid coding for both TlpA and eYFP](https://static.igem.wiki/teams/5154/sbol-design2.png)

Biorender SBOL design of cycle 2 plasmid design

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

![plates with no fluorescence](https://static.igem.wiki/teams/5154/engineering/cycle3/test2-tci-45c.jpeg)
![](https://static.igem.wiki/teams/5154/engineering/cycle3/test2-tlpa-45c.jpeg)

Left to right: Plate with TcI cells and plate with TlpA cells. Neither plates had fluorescent cells after being
placed on the hot plate at 45℃ for 5h.

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.

### Cycle 3: Two plasmid system with constitutive promoter 1

#### Design #3

![SBOL diagram of 2 plasmids, one coding for TcI, another for mRFP1](https://static.igem.wiki/teams/5154/engineering/cycle3/design3-sbol.png)

Biorender SBOL design of cycle 3 plasmid design

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).

![Gel electrophoresis of colony PCR of BL21 1p and 1p V1](https://static.igem.wiki/teams/5154/engineering/cycle3/240923-bl21-alpha-beta-1p-colony-pcr-labelled.jpg)
![Gel electrophoresis of colony PCR of 2p](https://static.igem.wiki/teams/5154/engineering/cycle3/2p-pcr-labelled.jpg)

Left top row: Colony PCR of 1p from BL21 cells. The bands appear around 700bp, and the expected band in all 4
samples is 953bp. Left bottom row: Colony PCR of 1p from DH5α cells. The bands appear around 700bp, and the
expected band in all 4 samples is 953bp. Right: Colony PCR of 1p V2 from DH5α cells. The bands appear around
1000bp, and the expected band in all 6 samples is 1041bp.

#### 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.

![Image of plate after 5h on the hotplate.](https://static.igem.wiki/teams/5154/engineering/cycle3/test3-hot-plate-testing.png)

Hot plate testing, cells remained white after 5h.

![2 plates of cells](https://static.igem.wiki/teams/5154/engineering/cycle3/test3-42c-incubator-test1.jpg)

Cells before and after 5h of hot plate testing and 1h in the 42℃ incubator. After growth overnight in the 37℃
incubator, the cells 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](https://2018.igem.org/Team%3ARDFZ-China), 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).

![Table of colours of samples](https://static.igem.wiki/teams/5154/engineering/cycle3/test3-table.png)

Plates were made with the same samples above and incubated at 24h at 4 different temperatures: 23℃
(room temperature in our lab), 30℃, 37℃ and 42℃.

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.

![plates afetr 24h incubation at different temperatures](https://static.igem.wiki/teams/5154/engineering/cycle3/test3-incubator-24h.jpg)

Plates with TcI BL21 cells after incubation at different temperatures. The 30℃ did not seem to have many
colonies after 24h, and all samples on the α plate that grew in 37℃ for 24h were pink.

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.

![](https://static.igem.wiki/teams/5154/engineering/cycle3/tci-ab-plates-after-48h.png)

Plates after 48h incubation

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)

![Plate reader data for fluorescence](https://static.igem.wiki/teams/5154/engineering/cycle3/test3-plate-reader-data.jpg)

Plate reader data of the relative fluorescence of cells after 24h of growth in liquid at different
temperatures.

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℃.

### Cycle 4: Two plasmid system with constitutive promoter 2

#### Design #4

![SBOL diagram of plasmid coding for both TlpA and eYFP](https://static.igem.wiki/teams/5154/engineering/cycle3/design4-sbol.png)

Biorender SBOL design of cycle 4 plasmid design

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.

![Gel electrophoresis of 1p V3 and 2p V1 and 2](https://static.igem.wiki/teams/5154/engineering/cycle3/tci-build-4-1p-2p-colony-pcr-labelled.jpg)
![](https://static.igem.wiki/teams/5154/engineering/cycle4/screenshot-2024-10-02-011500.png)

Left: Colony PCR of 1p V3 from DH5α cells. The bands appear around 1000bp, and the expected band in all 6
samples is 966bp. Right: Colony PCR of 1p V2 from DH5α cells. The bands appear around 1000bp, and the expected
band in all 6 samples is 1041bp. Bottom: Colony PCR of cells from build #4.

#### 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.

![4 petri dishes with 8 colonies each. Each plate shows different levels of fluorescence, and they were kept in different colonies](https://static.igem.wiki/teams/5154/engineering/cycle4/petri-dishes.png)

Starting from the bright red colony, going clockwise, alpha 9, pTi, 1A, 1B, 2A, 2B, 3A, and 3B. 3A
shows no visible fluorescence at 23C or 30C, but a distinct colour is noticeable in the centre of the colony at
37C and 42C

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.

![bar chart of plate reader data](https://static.igem.wiki/teams/5154/engineering/cycle4/bar-charts-withoutpti.png)
![bar chart of plate reader data](https://static.igem.wiki/teams/5154/engineering/cycle4/pti-bar-chart.png)

Plate reader data for fluorescence readings normalised to OD600 after 30 hours incubation at different
temperature

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).

![plate reader data for normalised fluorescence over time](https://static.igem.wiki/teams/5154/engineering/cycle4/samples.png)
![plate reader data for normalised fluorescence of the controls over time, as well as the change in temperature over the course of the experiment](https://static.igem.wiki/teams/5154/engineering/cycle4/controls.png)

Plate reader data for fluorescence readings normalised to OD600 over time as temperature increases

### Cycle 5: Two plasmid system with inducible promoter

#### 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).

![transformation of Bl21 in cycle 5](https://static.igem.wiki/teams/5154/engineering/cycle3/design5-biorender-transparentbg.png)

Biorender diagram of the 2 variations of design 5 that were built

#### 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.

![colonies on a plate](https://static.igem.wiki/teams/5154/engineering/cycle3/test5-new-67h-after-transformation.jpg)
![plate ith one colony](https://static.igem.wiki/teams/5154/engineering/cycle3/test5-old-67h-after-transformation.jpg)

![white cells on a yellow fluorescent agar plate](https://static.igem.wiki/teams/5154/engineering/cycle3/test5-new-48h-in-42c.jpg)
![white cells ith pink centres on a yellow fluorescent agar plate](https://static.igem.wiki/teams/5154/engineering/cycle3/test5-old-48h-in-42c.jpg)

Top left: Many colonies grew after 67h at room temperature. Top right: Only one colony grew after 67h at room
temperature, however more colonies gre once the plate was placed into the 42℃ incubator. Bottom left: All
colonies on the plate were white, the yellow colouration is due to the fluorescence of the LB agar. Bottom
right: The centre of the colonies as well as colonies on the edge of the plate are pink.

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.

### Cycle 6: Improvements of the one plasmid system

Note: While primers were designed and ordered for these cycles, we ultimately did not have time to carry out the
build/test/learn phases. Details are included here for future teams who might want to build on our previous work.

#### 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.

### Conclusion

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.

## Mechanical

#### Intro

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.

### Cycle 1: Paper replication

#### 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.

### Cycle 2: Transformation & blue-colony test

#### 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.

![Y100H transformed](https://static.igem.wiki/teams/5154/engineering/wsc1/image22.jpg)

We successfully Transformed Y100H growing on triple dropout media

#### 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.

![Master plate testing](https://static.igem.wiki/teams/5154/engineering/wsc1/image23.jpg)

(a) Masterplates incubated at 37°C overnight. 5, 3, 1 µl of X-gal is added to each
colony in each row, from left to right.

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.

### Cycle 3: Further investigation on blue-colony test

#### 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.

![30°C over the weekend](https://static.igem.wiki/teams/5154/engineering/wsc1/image51.jpg)
![37°C over the weekend](https://static.igem.wiki/teams/5154/engineering/wsc1/image24.jpg)

YPD masterplate testing with X-gal at different incubation temperatures. (a) X-gal added
after over-weekend incubation at 30°C. (b) X-gal added after over-weekend incubation at 37°C.

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.

![30°C overnight](https://static.igem.wiki/teams/5154/engineering/wsc1/image19.jpg)
![37°C overnight](https://static.igem.wiki/teams/5154/engineering/wsc1/image13.jpg)

![30°C, 2 nights](https://static.igem.wiki/teams/5154/engineering/wsc1/image48.jpg)
![37°C, 2 nights](https://static.igem.wiki/teams/5154/engineering/wsc1/image28.jpg)

Masterplates incubated at different temperatures for different time periods. (a).1: 30°C
overnight. (b).1: 37°C overnight. (a).2: 30°C, 2 nights. (b).2: 37°C, 2 nights. Result showed that for
2-nights incubation, 30 Celsius will have more activation than 37 Celsius.

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.

![30°C trial activation](https://static.igem.wiki/teams/5154/engineering/wsc1/image11.jpg)
![37°C trial activation](https://static.igem.wiki/teams/5154/engineering/wsc1/image25.jpg)

Blue colonies formed for both HAS100L and Y100H. Incubation at 30°C for 8 days to hydrolyze all
X-gal. (b) is the replication of (a) with the same experiment setting.

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.

![Liquid-based method for X-gal testing.](https://static.igem.wiki/teams/5154/engineering/wsc1/image31.jpg)

Liquid-based method for X-gal testing.

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.

### Cycle 4: MNPs binding and Magnetic Activation

#### 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.

![](https://static.igem.wiki/teams/5154/engineering/wsc1/image56.jpg)

250 nm MNPs viewed under the microscope.

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.

![MNPs_1](https://static.igem.wiki/teams/5154/engineering/wsc1/image16.png)
![MNPs_2](https://static.igem.wiki/teams/5154/engineering/wsc1/image37.png)

(a) (b): MNPs bound with the yeast cell through Ni-His tag interaction.

**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:

![platereader set](https://static.igem.wiki/teams/5154/engineering/wsc1/image63.png)

Plate reader setup for *S. cerevisiae* growth and toxicity assay.

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.

![growth](https://static.igem.wiki/teams/5154/engineering/wsc1/image6.png)

Growth curve of Y1 / HAS100L / Y100H over 48 hours. The result shows that Y1 has the
highest growth rate in YPD, followed by Y100H, with HAS100L showing the lowest growth rate.

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

![toxicity](https://static.igem.wiki/teams/5154/engineering/wsc1/image40.png)

Growth curve of Y100H incubated with different concentrations of 250 nm MNPs. The result
shows that 250 nm MNPs have negligible toxicity to *S. cerevisiae* Y100H over 48 hours.

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.

![1hr](https://static.igem.wiki/teams/5154/engineering/wsc1/image38.jpg)
![overnight](https://static.igem.wiki/teams/5154/engineering/wsc1/image10.jpg)
![3 nights](https://static.igem.wiki/teams/5154/engineering/wsc1/image8.jpg)

Effect of incubation time on color change after magnetic field activation. Plates are
incubated at room temperature after being activated for 1 hour. (a) Immediately after 1-hour activation; (b) One
night after activation; (c) 3 days after activation.

#### 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.

### Conclusion

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).

![](https://static.igem.wiki/teams/5154/engineering/wsc1/image21.png)

Growth curve of Y100H incubated with different concentrations of 250 nm MNPs. The result
shows that 250 nm MNPs have negligible toxicity to *S. cerevisiae* Y100H over 48 hours.

## Hardware

### Introduction

Commercial magnetogenetics hardware is expensive and inflexible so we built our own. First we set about
designing, building and testing specific electromagnet designs for targetting each of our magneto-receptive
pathways. Then we developed a research platform to extend our project allowing for precision spatial targetting
magnetogenetics, called MagentaBox, to make experimentation repeatable and replicable.

### Cycle 1 - High Frequency Coil Design

#### 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](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27hardware%27%29%20%7D%7D)). 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:

![Coil 1](https://static.igem.wiki/teams/5154/hardware/coil1.png)

#### Coil V1

* Turns: 5
* Inductance: 1.72 \(\mu H\)
* Oscillation frequency: 365kHz
* Peak Field Strength: N/A

Notes: unable to test heating efficacy due to lack of support structure.

![Coil 2](https://static.igem.wiki/teams/5154/hardware/coil2.png)

#### Coil V2

* Turns: 5
* Inductance: 1.57 \(\mu H\)
* Oscillation frequency: 382kHz
* Peak Field Strength: 0.58mT

Notes: added support structure and tested initial heating efficacy however it was found to be low.
Decided to increase power of driver circuit and scale to small size first to confirm heating was
possible.

![Coil 3](https://static.igem.wiki/teams/5154/hardware/coil3.jpg)

#### Coil V3

* Turns: 6
* Inductance: 0.41 \(\mu H\)
* Oscillation frequency: 985kHz
* Peak Field Strength: N/A

Notes: oscillation drive frequency proved too high for driver circuit and one of the MOSFETs broke.
Decided to increase the number of turns to increase the inductance and thus reduce the oscillation
frequency.

![Coil 4](https://static.igem.wiki/teams/5154/hardware/coil4.jpg)

#### Coil V4

* Turns: 12
* Inductance: 1.93 \(\mu H\)
* Oscillation frequency: 534kHz
* Peak Field Strength: 12.5mT

Notes: At this field strength and frequency it was possible to achieve high heating efficacy in the
small volume which helped us learn a lot about our MNP targets. We then looked to scale up our design
back to the size of a petri dish.

![Coil 5](https://static.igem.wiki/teams/5154/hardware/coil5.jpg)

#### Coil V5

* Turns: 5
* Inductance: 0.94 \(\mu H\)
* Oscillation frequency: 493kHz
* Peak Field Strength: 2.11mT

Notes: Applying what we learned from the small scale designs and some finite element analysis we were
able to generate a significantly higher power coil for our target volume. The fundamental limitation
that remained was the Litz wire which had a low current limit.

![Coil 6](https://static.igem.wiki/teams/5154/hardware/coil6.jpg)

#### Coil V6

* Turns: 2
* Inductance: 0.147 \(\mu H\)
* Oscillation frequency: 654kHz
* Peak Field Strength: 4.71mT

Notes: This design used copper tubing which is the other common material for high frequency induction
coils and can be run at higher currents. The tradeoff is higher thermal losses due to skin effects.
This coil was not as effective at heating as V4 but performed significantly better than V5 due to the
material change.

![Coil 7](https://static.igem.wiki/teams/5154/hardware/coil7.jpg)

#### Coil V7

* Turns: 3
* Inductance: 0.31 \(\mu H\)
* Oscillation frequency: 450kHz
* Peak Field Strength: 3.81mT

Notes: Finally we developed an oval shaped coil designed to work as part of our final spatial
targetting system that could fit around our target dish.

❮
❯

#### 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.

![Comparing MNP heating efficacy in Coil V4](https://static.igem.wiki/teams/5154/hardware/comparing-mnps.png)

Different MNP heating in coil V4

![Comparing heating efficacy of EFH1 in different coils](https://static.igem.wiki/teams/5154/hardware/comparing-coils.png)

PEFH1 heating in different coils

![Comparing heating efficacy of EFH1 at different coil currents](https://static.igem.wiki/teams/5154/hardware/comparing-currents.png)

EFH1 heating with different input currents

#### 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.

### Cycle 2 - Low Frequency Coil Design

#### 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.

![Box diagram of system](https://static.igem.wiki/teams/5154/hardware/block-diagram.png)

Box diagram of the system

#### Build #2

We then build this design:

![Whole system](https://static.igem.wiki/teams/5154/hardware/full-setup.jpg)

System Build

For more information on this build, see our [hardware page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27hardware%27%29%20%7D%7D).

#### Test #2

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

![Yeast on coil](https://static.igem.wiki/teams/5154/hardware/yeast-setup.jpg)

Wsc1 Test Setup

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.

### Cycle 3 - MagentaBox V1

#### 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](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27hardware%27%29%20%7D%7D).)

#### Build #3

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

![Parts for MagentaBOX V1](https://static.igem.wiki/teams/5154/hardware/beforeassemblya.jpg)

Parts for MagentaBOX V1

![Assembled MagentaBOX V1](https://static.igem.wiki/teams/5154/hardware/afterassemblya.jpg)

Assembled MagentaBOX V1

#### 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.

![Demoing the device](https://static.igem.wiki/teams/5154/humanpractices-dhriti/humanpractices-makespacephotos2.png)

User Testing

#### 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.

### Cycle 4 - MagentaBox V2

#### 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.

![V2 CAD Design](https://static.igem.wiki/teams/5154/hardware/boxdesign.png)

V2 CAD Design

#### Build #4

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

![Internals MagentaBOX V2](https://static.igem.wiki/teams/5154/hardware/deviceb.jpg)

Device Build

#### Test #4

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

![Fluorescent Imaging MagentaBOX V2](https://static.igem.wiki/teams/5154/hardware/devicec.jpg)

Device in operation

![Fluoresence Calibration](https://static.igem.wiki/teams/5154/measurement/calibration-7-edited.png)

#### 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.

### Conclusion

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.

## References

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.

Illustrations created in BioRender.com


#### Test #2

In the second round of test, we successfully transform Y1 with pHPS100H to get the new strain we need for magnetic activation.

![Y100H transformed](https://static.igem.wiki/teams/5154/engineering/wsc1/image22.jpg)

Transformed Y100H growing on triple dropout media

To verify our construct, we incubated three strains (Y1,HAS100L,Y100H) at 37 Celsius overnight to activate their CWI pathway by high incubation temperature. Y1 does not carry lacZ reporter so was used as a negative control, while HAS100L contains lacZ reporter and used as a positive control in the test. ALl colonies were treated with X-gal after overnight incubation

![Master plate testing](https://static.igem.wiki/teams/5154/engineering/wsc1/image23.jpg)

(a) Masterplates incubated at 37°C overnight. 5, 3, 1 µl of X-gal is added to each
colony in each row, from left to right.

The result suggested that the construct is valid the transformation is successful.

#### Test #3

In the third round of test, we further investigated on the conditions of CWI pathway activation. This is to avoid getting a false positive result and incorrect conclusion in the following magnetic activation experiment.

We first tested the effect of incubation time / temperature on CWI pathway activation

![30°C overnight](https://static.igem.wiki/teams/5154/engineering/wsc1/image19.jpg)
![37°C overnight](https://static.igem.wiki/teams/5154/engineering/wsc1/image13.jpg)

![30°C, 2 nights](https://static.igem.wiki/teams/5154/engineering/wsc1/image48.jpg)
![37°C, 2 nights](https://static.igem.wiki/teams/5154/engineering/wsc1/image28.jpg)

Masterplates incubated at different temperatures for different time periods. (a).1: 30°C
overnight. (b).1: 37°C overnight. (a).2: 30°C, 2 nights. (b).2: 37°C, 2 nights. Result showed that for 2-nights incubation, 30 Celsius will have more activation than 37 Celsius.

Another test showed that for over-weekend incubation, 30 Celsius also showed more activation than 37 Celsius

![30°C over the weekend](https://static.igem.wiki/teams/5154/engineering/wsc1/image51.jpg)
![37°C over the weekend](https://static.igem.wiki/teams/5154/engineering/wsc1/image24.jpg)

YPD masterplate testing with X-gal at different incubation temperatures. (a) X-gal added
after over-weekend incubation at 30°C. (b) X-gal added after over-weekend incubation at 37°C.

The result showed that for long-term incubation, 30 Celsius will have more CWI pathway activation than 37 Celsius. 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.

![30°C trial activation](https://static.igem.wiki/teams/5154/engineering/wsc1/image11.jpg)
![37°C trial activation](https://static.igem.wiki/teams/5154/engineering/wsc1/image25.jpg)

Blue colonies formed for both HAS100L and Y100H. Incubation at 30°C for 8 days to hydrolyze all X-gal. (b) is the replication of (a) with the same experiment setting.

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.

![Liquid-based method for X-gal testing.](https://static.igem.wiki/teams/5154/engineering/wsc1/image31.jpg)

Liquid-based method for X-gal testing.

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.

#### Test #4

In the fourth round of test, we viewed 250 nm MNPs attached to yeast cells under a light microscope (objective 100\*).

![MNPs_1](https://static.igem.wiki/teams/5154/engineering/wsc1/image16.png)
![MNPs_2](https://static.igem.wiki/teams/5154/engineering/wsc1/image37.png)

(a) (b): MNPs bound with the yeast cell through Ni-His tag interaction.

**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:

![platereader set](https://static.igem.wiki/teams/5154/engineering/wsc1/image63.png)

Plate reader setup for *S. cerevisiae* growth and toxicity assay.

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

![growth](https://static.igem.wiki/teams/5154/engineering/wsc1/image6.png)

Growth curve of Y1 / HAS100L / Y100H over 48 hours. The result shows that Y1 has the
highest growth rate in YPD, followed by Y100H, with HAS100L showing the lowest growth rate.

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

![toxicity](https://static.igem.wiki/teams/5154/engineering/wsc1/image40.png)

Growth curve of Y100H incubated with different concentrations of 250 nm MNPs. The result
shows that 250 nm MNPs have negligible toxicity to *S. cerevisiae* Y100H over 48 hours.

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.

![1hr](https://static.igem.wiki/teams/5154/engineering/wsc1/image38.jpg)
![overnight](https://static.igem.wiki/teams/5154/engineering/wsc1/image10.jpg)
![3 nights](https://static.igem.wiki/teams/5154/engineering/wsc1/image8.jpg)

Effect of incubation time on color change after magnetic field activation. Plates are
incubated at room temperature after being activated for 1 hour. (a) Immediately after 1-hour activation; (b) One
night after activation; (c) 3 days after activation.