Results

To see how our key results fit into our proof of concept, please see the proof of concept page.
Each of the tests are different cycles of the DBTL cycle. To see the full DBTL cycles on the engineering page.

Test #1:

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

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.

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.

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.

Test #5:

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

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

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

Cloning

Cycle 1:Assembly unsuccessful, as cells were unable to grow on antibiotic plates

Cycle 2:Transformed cells were able to grow on LB+Amp plates, but sequencing of the plasmid suggests mistakes. Importantly, there seem to be issues with the terminator downstream of the TlpA coding sequence

Cycle 3 and 4:Cells grow on Chl + Kan plates but colony PCR suggests assembly is incomplete, as the insert containing the TcI transcriptional unit is ~200bp shorter than it should be. This suggests that it is missing one or more of the following components: promoter, ribosome binding site, or terminator.

Cycle 5: Cells grow on Amp + Kan plates, we did not have time for further verification

Test #2: Hot plate testing

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

Test #3: Hot plate and incubator testing

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

Following this test, we wanted to see if expression of mRFP1 could be increased by increasing the incubation time. After reading RDFZ-China 2018 iGEM team’s wiki page, we decided to incubate different colonies off the same plate for 24h.
6 colonies from each plate (α and β) were inoculated in liquid as well as plated on solid double antibiotic plates over the weekend at room temperature. After 65h, the colour of the cells were mostly white with some samples either with a pink centre or were fully pink (see table 3).

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 _). These plates suggested that temperature did have an effect on the expression of mRFP1.

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

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

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

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

Test #4: Incubator testing

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

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

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

Test #5: Incubator testing

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

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

Test #2

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

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

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

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

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.

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

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

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

Test #4

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:

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

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

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

MNP suspension and toxicity in agar

We tested different concentrations and methods of incorporating ferrofluid (first4magnets EFH1 F4MFF20-20) into agar and we wanted to test if:

Could we suspend the oil based ferrofluid in less oil and more water - yes

Could the E. coli cells survive on oil based ferrofluid and washed ferrofluid in agar - yes

Could we heat up the ferrofluid agar plates using our magnet system - no

Could we heat up the ferrofluid in a petri dish using our magnet system - yes

Test #1: Could we suspend oil based ferrofluid in more water - yes

We wanted to suspend the ferrofluid in water as we were concerned the oil may kill or prevent further growth of E. coli cells.

After many different approaches (see Wet lab notebook pt 2 ‘MNP dispersion and agar testing Thursday 12/09/24’), we designed a protocol to wash the oil off of the magnetic nanoparticles (See custom protocols: MNP (EFH1 Ferrofluid) wash and suspension in water).

We were successful in washing, mixing and pouring LB agar plates mixed with ferrofluid. After washing, there was visibly more even suspension of the MNPs within the agar than compared to without washing (see below). These were 35mm petri dishes.

We repeated many different variations of plates including:

LB agar control with no ferrofluid

Ferrofluid directly into the agar

Ferrofluid directly in water in different ratios into agar

Washed ferrofluid at different concentrations into agar

Ferrofluid directly into the agar with a layer of normal agar on top

The concentrations ranged from 0 to 3.2 mg/mL.

Test #2: Could the E. coli cells survive on ferrofluid in agar - yes

Surprisingly, all plates grew cells overnight at 37℃, even cells that were in direct contact with the ferrofluid.

Therefore, we next tested if the plates could heat up within our coil design.

Test #3: Could we heat up the ferrofluid agar plates using our magnet system - no

After the cells grew, we placed the plates onto the magnetic coil for 10 minutes, and checked the temperature using the thermal camera imaging station we created. Unfortunately, none of the plates heated up to a temperature high enough to heat shock any of the cells (~37-42℃).

Therefore we wanted to work out if the issue lie in the hardware design or the low concentration of MNP in the agar. Our next step was to test if the ferrofluid itself could be heated up using the hardware.

Test #4: Could we heat up the ferrofluid in a petri dish using our magnet system - yes

We previously ran an experiment demonstrating that ferrofluid could be heated up to higher temperatures in a PCR tube. However, we wanted to be certain that this was reproducible in a 35mm petri dish.

3ml of ferrofluid (EFH1, 100mg/ml) was poured into a 35mm petri dish. The starting thermal image was taken. Coil V7 was applied around a segment of coil for 2 minutes, and the thermal image was taken again. Water was used as a negative control.

This demonstrated that the ferrofluid could indeed be heated up inside of a 35mm petri dish using our magnet system. Therefore, the next step was to increase the concentration of ferrofluid in the agar and run both growth and heat tests (test #2 and #3).

Conclusion

As a proof of concept, untransformed BL21 E. coli cells can grow on ferrofluid agar plates, and the ferrofluid can be heated up by our magnet system. With more time, we would have:

Repeated tests #2 (toxicity and growth) and #3 (heating) with higher ferrofluid concentrations

Repeated test #2 with transformed BL21 E. coli cells to see if the burden imposed by transformation made them more sensitive to magnetic nanoparticles