iGEM 2022 — Team sheffield

{% extends "layout.html" %} {% block title %}Notebook{% endblock %} {% block
lead %}A notebook to keep track of progress{% endblock %} {% block page\_content
%}

Here we have outlined all of our wet and dry lab experiments over the course
of the project. Here we design a modular, plasmid-based toolkit for
continuous directed evolution and a DIY bioreactor for automated continuous
culture.

###### **March**

In March we decided to recap the team’s biology knowledge and familiarise
members with central dogma of biology (cloning, plasmids, etc). We collated
funding from all of the different departments that our team represented as
well as that from external sponsors (JMP). During our brainstorming sessions,
each team member presented a problem and potential solution for different
topics that we could potentially take up as an iGEM project. We also planned
how we would organise our daily notebook diaries throughout the project: we
decided to use Github, Benchling, Google Docs, Research Rabbit, and Zotero.

###### **April**

To practice fundamental microbiology techniques and familiarise non-biology
team members with the lab, we decided that amplifying DNA using
*E. coli* was a good place to start. We used DNA from 2019’s iGEM
distribution kit and amplified them by transforming into bacterial cells. We
also completed our team’s registration for the Grand Jamboree.

###### **May**

Unfortunately May was exam period at the University of Sheffield and final
projects were being submitted. In the meantime, team members focused on
researching the shortlisted ideas before we decided on the final project in
our next meeting.

###### **June**

Our first team meeting with our PI - Professor Tuck Seng Wong - was held which
resulted in a slight change to the project’s initial scope in order to be more
achievable within the iGEM timeframe. He suggested using the
**MutaT7 system** for cellular in vivo continuous directed evolution to
evolve a prokaryotic β1–4 Galactosyltransferase for increased expression and
lactose production. We looked into identifying a lactose biosensor to tie
fitness to improved lactose production. Professor Tuck Seng Wong also
suggested exploring the lac repressor while designing our genetic circuit for
selection. The Mutat7 system could be applied to other iGEM teams’ parts
during collaborations. We also discussed using JMP’s sponsorship software to
optimise VMax Express growth and expression even further. We discussed using
the HiSCRIBE system as a hypermutation mechanism - to increase error rate by
deleting a proofreading domain and never before published mechanism for
directed evolution

We worked through setting up some *E. coli* overnight cultures,
experiencing issues with aseptic technique and use of cells in solution rather
than using plate colonies. We learnt from this however and can improve for the
next overnight culturing session. Using the overnight cultures, we worked on
building some *E. coli* growth curves by measuring incubated samples'
optical density every 20 minutes over 3 hours. Ideally it would have run until
the growth plateaued (considered when 3 consecutive readings show no change),
but there was not enough time. However, enough data was collected to
approximate a doubling time for growth.

#### **Start of lab work!**

###### **Week 1**

Week 1 of July consisted of meetings with our PIs from engineering departments
to discuss whether our ideas for the shape of the bioreactor were suitable and
to further gain advice regarding the use of turbidostats. We also submitted
the impact grant application and were very excited to hear the results in due
course. Planning the team dynamic was a crucial part at this stage in the
project; we assigned subteam leaders to give people directionality and ensure
all team members were working in tandem.

We started investigating genetic parts we would like to work with. This
included the MutaT7 system (Paris Saclay 2021, Alvarez, Moore), the HiSCRIBE
system, and various growth-based selection mechanisms. As well as this we
began designing proof of concept assays (e.g. silent ATG start codons).

On the dry lab side of things we began familiarising ourselves with various
software (e.g. Fusion 360) for 3D modelling of the bioreactor. We started
coding bioreactor parts and our engineers sketched first drafts of the
toroidal bioreactor and ordered parts.

In the lab we completed growth curves for *V. natriegens* for media
optimization.

###### **Week 2**

This week we performed lots of growth curves and eliminated candidate
ingredients that negatively impacted *V. natriegens* growth to aid media
optimisation.

We got in touch with the makers of HiSCRIBE to get plasmid maps to work with,
as well as emailing our sponsor JMP to get details on biochemical pathway
optimization.

We also identified lots of growth slowing genes to design our selection
plasmid, and sourced plasmid backbones as well as finalising the design of the
MutaT7 plasmid. We decided the lac promoter we hoped to use was too leaky for
our selection mechanism to work (risk of evolutionary cheaters) so decided to
improve the lac promoter to contribute to part improvement. We also designed
our test casset for MutaT7

We tested the Raspberry Pi with the code we had written and built a prototype
of our bioreactor to model fluids and heating components. We ordered
components for the stirring mechanism to test in week 3.

###### **Week 3**

We decided that a phototransistor was the best option for OD measurements. We
explored the phototransistor and LED pair components to do refraction
modelling to see how the refractive index of perspex would affect the rays of
light from the LED - some LED pairs caused internal refraction along with the
bioreactor walls. Looking into finding LEDs with narrower range and
transistors that are highly sensitive. Then, we continued with modelling with
LEDs and transistor pairings.

###### **Week 4**

In the lab, we finished our growth curves with e2yt as well as testing
*V. natriegens* growth on sorbitol. We also mini prepped our backbones
(pUC18/19). Arjun generated recipe cards for media using his design of
experiments model in the JMP software to perform more growth curves to hone in
on the optimal media for *V. natriegens*.

We finished designing the MutaT7 test cassette and MutaT7 fusions. Going to
take a swing at ordering the sequences today in g-blocks as well as ordering
anhydrotetracycline (aTc) inducer for fusion plasmid.

Chalisa did some modelling for oxygen diffusion in the bioreactor. We ordered
more parts and found a perfect ceramic sealant for our bioreactor that would
fix leakages, create more turbulence thus leading to better mixing of the
media and also manipulate the bioreactor into a dish bottom shape. We also
connected the peristaltic pump to the breadboard. Worked fine, but heated up
when connected to the motor controller. Possibly short-circuited somewhere.

We also collaborated with Ohio State University. We wanted to hear their
insights into how they modelled their own bioreactors virtually. Our coding
member Kesler shared some useful python code for a system of ODEs that could
help their team. We hope to receive a useful set of growth modelling codes
from OSU for our experimental data and approximate rate constants for
*V. natriegens* that could be substituted in.

###### **Week 5**

Media optimisation experiments continued with more media versions being made
up by several lab members. It was found that Na2HPO4 and MgCl2 forms an
insoluble magnesium phosphate precipitation when both are added in media, but
a work around was constructed by Arjun through swapping MgCl2 for MgSO4 and
replacing Na2HPO4 for more CaCl2. We failed to transform
*V. natriegens* with pUC18/19, but the *E. coli* transformations
worked. We will remake competent *V. natriegens* to see if that is the
issue. We fixed our main issue with our media that kept precipitating by
removing NaPO4.

For the bioreactor we: gathered the equipment; connected the L239D to the
breadboard; connected the peristaltic pump, motor and raspberry pi pico;
established micropython code and hardware set up; filled the prototype with
water and pump water in a cycle and measured flow rate. The pump speed and
flow rate was suspiciously low- we realised something was short of circuit due
to the power supply overheating quickly. We also identified a small leakage in
the bioreactor prototype which we needed to prioritise by sealing with more
acrylic cement. We also built a second prototype to test stirring using
magnetic balls and magnetic rods.

###### **Week 6**

This week we recorded our promotional video! :)

Our *V. natriegens* transformations are still not working well. We got a
few colonies to miniprep and sequence.

We also miniprepped a bunch of plasmid backbones:

* pET21a - AmpR
* pBBRBB - KanR
* pBBRBB - SmR
* pET28a - KanR
* pBR52b - AmpR
* pACYDuet - CmR

For the bioreactor, we worked on the peristaltic pump. Solder wires onto the
peristaltic pump to secure it, divert the wire from the power supply (so we
can supply the breadboard with 5V and 12V) and follow the hardware setup and
code. We measured the **volumetric flow rate (= 60mL/min)** and checked
voltage across different connections using a multimeter (10V between motor
connections, 12V supplying L293D showing power loss within the circuit).
Moving forward we will make sure **Raspberry Pi Pico** is powered by 5V
(not 12V), there are no unwanted wires disconnected from the breadboard but
this may accidentally short the circuit, switch off the circuit before
changing any connections. Resistance caused around 40% loss in power supply to
the motor, so the voltage supplied is only 10V - why the volumetric flow rate
is only 60mL/min. While checking the voltage, current and power between
different points in the circuit, the Raspberry Pi blew up (most likely
accidentally connected the Raspberry Pi to 12V instead of 5V). After this we
built our third bioreactor prototype and it was concluded that a fourth
prototype needed to be created- one with refined lids and deeper/wider base
etchings.

###### **Week 7**

This week we acquired CRISPRi plasmid from our university department. We also
completed the travel Risk assessment for Paris and Munster. We designed the
layout of our poster and presentation and planned our promotional video.

In the lab, we planned an experiment for baseline activity of GalT and a
cloning strategy for RNA attenuator as well as planning out final media
optimisation experiments (Arjun)

We then recorded all of the bioreactor’s prototype journey so far!

###### **Week 8**

We decided controlling Pump Power with Potentiometer was a good idea to ensure
there was a way to turn the pump off completely and we tried to figure out
quantitative value for speed for the Servo Motor. For the OD system, the
original code reported the OD as transmittance rather than absorbance so we
modified it to 100-light so that it measures absorbance instead. We addressed
the outflow port issue, implemented a new downward tube to overcome surface
tension issues outlined in the Engineering Success page. We finalised the
design for the temperature sensor on the screen of the bioreactor.

Some of our team attended the European meetup in Muenster, Germany where we
gained and received feedback about our posters, presentation delivery and
overall project! We also made

###### **Week 9**

More bioreactor testing, more growth curves (but now done in baffled flasks).
We used HiFi assembly to generate all of our constructs: the MutaT7 test
cassette, MutaT7 base editor fusions, growth slowing genes/ SDH under the
control of a lac promoter and our part improvement (improved Lac promoter
controlling the expression of GFP so we can compare the strength of the
original promoter, to our improved promoter). Upon inducing the lac promoters
we found no significant expression - maybe our IPTG induction is not working.
We sent off our constructs for sequencing to confirm we constructed the
correct things. Restriction digests confirmed our assemblies were correct.

###### **Week 10**

Wrapped up experiments and moved the team's complete focus to the Wiki page.
Our team collaborated with Women in Engineering (WiE) Society for an outreach
session to Arbourthorne Community Primary School. The session consisted of
modelling DNA using plastic beads that were transformed into keychains for the
children to take home.

{% endblock %}

{% extends "layout.html" %} {% block title %}Engineering Success{% endblock %}
{% block lead %}We encountered many issues throughout our project, so it was
only right that we solved them! Here you can find out about our engineering
successes{% endblock %} {% block page\_content %}

#### **Media Optimisation Engineering Success**

###### **Introduction**

We demonstrated engineering success while optimising our media for
*V. natriegens*. A fast *V. natriegens* growth rate is important
for our project as our modelling has shown that a faster growth rate
should lead to a lower fixation time, which would make our evolutions more
efficient. Since we are optimising our media for growth rate, it is
important for our project that we do so in an efficient and sensible way.
The engineering cycle provides a way to do this. For a deeper dive into
our Design of Experiments guided media optimisation, see
[our dedicated page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27media-circus%27%29%20%7D%7D).

###### **Design (*in silico*)**

When we first chose ingredients to include in our test media recipes, we
focused on individual ingredients that looked beneficial for growing
*V. natriegens*, but we did not consider how they would react. The
purpose of this design was to efficiently screen through potentially
beneficial *V. natriegens* growth media ingredients, using a
statistical model whose output would be a linear model with the estimated
effect sizes and p-values (significance) of the ingredients on growth
rate. At this stage, we had not seen statistical design of experiments
used to systematically find an optimal growth media for
*V. natriegens* in the literature. We have seen similar methods used
to optimise media for other microorganisms.

###### **Build**

We then started making media as planned, a few at a time through the
\(25\) selected media recipes. We quickly found that some recipes (media
\(5\) and \(8\) in particular) were making insoluble precipitate. When we
encountered the problem, we stopped to perform diagnostic tests, to
identify the problem.

###### **Test**

We then tested mixing pairs of ingredients together to isolate ingredients
that were reacting. We were suspicious of the salts because the media with
precipitate had many of the salts in them. We added pairs of salts to a
beaker of distilled water and stirred. We were testing to see
qualitatively if there was any solid precipitate.

###### **Learn**

We found that \(\ce{MgCl2}\) and \(\ce{Na2HPO4}\) were reacting, which was
not anticipated. We have also learned here that it could be worth doing
some preliminary tests before even going into the screening experiment. To
move past this problem we replaced \(\ce{MgCl2}\) with \(\ce{MgSO4}\) and
\(\ce{Na2HPO4}\) with \(\ce{CaCl2}\). To avoid making the same mistakes
again, we repeated the test of mixing pairs of ingredients in beakers of
distilled water and found that now none of our media were reacting to form
precipitate.

###### **Application**

Learning from this, we decided to do a scoping experiment between the
screening and main experiments to both avoid wasting time and to maximise
the information gained in the main experiment. Following the screening
experiment, we saw that we should use more yeast, tryptone and
\(\ce{MgSO4}\). We wanted to know (**design**) how much more we could
use without saturating our media by putting too much in. We were wary
about this because of our previous problems with precipitate. To estimate
where the boundary of saturation was, we selected (**build**) a ‘high’
concentration media (H) and a ‘very high’ concentration media (VH). We
found (**test**) that VH media was too high and left undissolved
residue at the bottom of the duran bottle, but media H was fine.
Consequently (**learn**), we then used ingredient levels in media H as
our ‘high’ ingredient levels in our main experiment.

###### **Conclusion**

In the end, this led to us finding optimal levels of yeast, tryptone,
\(\ce{MgSO4}\), and NaCl in our final media, giving a doubling time of
\(15.6\) minutes, without doing an excessive number of experiments. The
engineering cycle helped by providing an effective way to identify and
move past unforseen problems we encountered along the way.

#### **Bioreactor Design Engineering Success**

Throughout the process of constructing our bioreactor, we had to move
through many iterations of the engineering DBTL cycle. For more
information on how we designed and built our toroidal bioreactor, see
[our dedicated page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27hardware%27%29%20%7D%7D).

###### **Main Vessel Design**

The main vessel we designed and then built is a square-sectioned toroid.
The walls are made from two perspex cylinders of differing diameters and
with 3mm thick walls. Both cylinders are cut down to 45mm, and the smaller
one is placed inside the larger one. A 5mm thick bottom plate is then used
to secure the two cylinders in place to ensure a constant distance between
the outer and inner walls. The initial bottom plate was an annulus cut
with a laser cutter. It had enough width to allow the inner cylinder to
sit on the inner edge, and the outer cylinder to sit on the outer edge.
Then the cylinders and annulus plate would be bonded with acrylic cement.
However, due to the frankly low-tech means of construction this resulted
in the cut cylinder sections not sitting perfectly flat on the plate. This
meant that acrylic cement could not bond all of the cylinders to the
bottom plate, leading to areas where the vessel would leak. Multiple
applications of cement were applied to “shore up” these holes, but this
resulted in a varied topography on the bottom of the plate. Moreover,
there was no lip in the middle of the toroid to place the servo stage on.
This also inadvertently resulted in the inner and outer cylinders not
being parallel, which caused magnetic balls to get caught between the
walls occasionally.

It became very apparent that we needed a much more precise method of
construction that was still intuitive and replicable. Our second bottom
plate was still an annulus but with a reduced inner diameter and increased
outer diameter. A laser cutter was used to create “grooves” for the
cylinders to slot into for three main reasons. Firstly, to keep a constant
distance between the vessel walls and prevent magnetic balls from getting
stuck. Secondly the grooves would maximise how much of the bottom plate
and the cylinder walls would be touching, so we would only have to use
minimal amounts of cement for maximum adherence and ensure the vessel was
completely watertight. Thirdly and finally, the vessel walls slotting into
the bottom and being chemically bonded increased the structural integrity
of the vessel overall.

The lid was designed with the same rough technique, but with two main
differences. Firstly, the grooves engraved were wider in order to prevent
a tight fit from occurring and allowing for moderate airflow. Secondly,
small holes were cut into it to allow for insertion of a temperature probe
and for the fluid input. This is because the original lid we had cut was
just a flat plate we intended to place on top, but this had the same
issues as the bottom plate: it left gaps between the walls and the lid,
which could result in contamination.

###### **Outflow of Spent Culture**

Initially, we thought that a simple hole drilled into the side of the
outer wall would be enough. The idea was that the bottom of the outflow
hole should line up with where the top of the culture volume would be.
Hence, any surplus volume would trickle out of the hole through a silicon
tube and into a waste beaker or some other vessel. However, it quickly
became apparent that the intended outflow hole was not large enough to
allow for easy flow once 100ml was reached; surface tension was causing
adhesion to the sides of the hole. With the hole along present as the
drainage method, surface tension holds approximately 20ml more volume than
intended before pressure from surplus culture builds enough to release
20+ml at once. Once a silicon tube was inserted to direct the flow into a
vessel, the decreased diameter of the outflow caused even more water to
build, and once pressure builds enough then as much as 40ml is released at
once. This is extremely problematic, as it is poorly controlled and with
seemingly inconsistent volume outflow episodes.

If we wanted to stick with the outlow hole idea we had two possible
solutions, each with possible drawbacks. Firstly we could simply make the
hole larger and larger until outflow occurred at the right volume, but
this could impact upon heat retention of the vessel and present a possible
method for contamination. Secondly, we could keep the hole diameter the
same but change the shape to be square. The idea behind this is to
minimise surface tension effects in the middle-most parts of the edges of
the square. However, a square hole is harder to reproduce for anyone
wanting to build our bioreactor. Moreover, square tubing is less commonly
used and we wanted to design our bioreactor with common, off the shelf
components.

Eventually we had the idea of inserting a rigid tube at an upwards angle,
where the top of the tube would sit at precisely where 100ml volume would
be (see below).

![](https://static.igem.wiki/teams/4451/wiki/engineering/screenshot-from-2022-10-12-07-29-17.png)

Figure 1: The improved outflow design of the bioreactor

This method proved to be much more reliable, as gravity alone is a strong
enough force to overcome surface tension issues. The bioreactor drains at
approximately 105-108ml instead of 140+ml like the old system, much more
controllable and conducive to continuous culturing. Moreover the angle of
the inserted tube is helpful in preventing airflow containing microbes
into the bioreactor, which lessens the likelihood of contamination.

###### **Peristaltic Pump for Influx of Media**

A peristaltic pump was a given for our modular design, being relatively
affordable and highly programmable. Initially, we chose a 6V pump, but it
would overheat the MB-102 breadboard’s 5V power supply and the Raspberry
Pi. Parts of the circuit were heated up to as much as 120°C, which was
obviously unsustainable and potentially dangerous. Moreover, we were only
reaching 12mL/min pumping rates with a microcontroller, and only
46.6mL/min when connected directly to the power supply. The advertised
maximum pumping rate was 100mL/min so something was clearly wrong.

Our first attempt to rectify this was swapping out the Raspberry Pi Pico
unit and double-checking that the circuit was correct. However, the
heating problem still persisted. We then thought it could have been the
MB-102 breadboard power supply and so instead powered 12V directly to the
breadboard instead. This did not solve the heating issue either.

On our third attempt, we measured the voltage across the power and ground
line separately and found that the breadboard was not actually separated
in the middle. We switched to a 12V pump and altered the circuit slightly
so as to pump it directly from the wall adapter via a separate miniature
breadboard, instead of the breadboard supply as the breadboard’s linear
regulators were turning all waste power into heat. This proved to be much
more effective in achieving advertised pumping benchmarks, and it stopped
the overheating problem as well.

###### **Magnetic Stirring System**

We had the idea of employing a magnetic stirring system using a bifurcated
horn underneath the toroidal vessel that had magnets attached to each end.
The inspiration behind this was simply the magnetic stirring systems
commonly found in many laboratories. The middle of the horn was attached
to an inverted servo to rotate 360°. These magnets would attract magnetic
balls inside the culture, and as the horn rotated the magnetic balls
inside the culture would be dragged along to agitate and mix the culture.

The first servo we purchased actually did not have 360° rotation and could
only rotate 180° in either direction. Hence we purchased another. We
initially found we could not control the direction and speed of the servo.
To remedy this, we calculated the number of ticks for the PWM driving the
servo (65535), the total time of one period (20ms), and the duty cycle
on-times for clockwise (1ms) and anticlockwise (2ms) as well as the stop
instruction (1.5ms). We composed the following formula to calculate the
correct number of ticks to get the desired duty-cycle and rotation
behaviour:

* For motor to turn clockwise: \(\dfrac{1}{20} \times 65535\)
* For motor to turn anticlockwise: \(\dfrac{2}{20} \times 65535\)
* For motor to stop: \(\dfrac{1.5}{20} \times 65535\)

From here we scaled them from -100 to +100 to make it easier to control.
This was calculated by dividing the number of active ticks with 20 and
multiplying by 65535 with the following code:

```
def servomove(power)
        pwm.duty – u16(int((power/100)*1638.75)+4915.125))
```

The values in the above code were derived in the following way:

* \(\dfrac{1}{20} \times 65535 = 3276.75\)
* \(\dfrac{1.5}{20} \times 65535 = 4915.125\)
* \(\dfrac{2}{20} \times 65535 = 6553.5\)
* Difference between each = 1638.375
* Median value = 4915.125

Once the scaling from -100 to +100 is applied, the equation simplifies to:

\[\dfrac{P}{100} \times 1638.375 + 4915.125\]

Where P is the power in (-100,100). Hence, if power is -100, then the
number of ticks input must be 3276.75 which corresponds to a clockwise
turn.

Originally, we were stirring unidirectionally as this was the most obvious
option for such a small volume. However, we began to suspect that this
resulted in laminar flow in parts of the bioreactor which was not suitable
for media mixing. Hence, we altered our code and instead adopted a
bidirectional stirring approach. Opting for a bidirectional stirring
system proved to be the right decision when we came to testing our
bioreactor mixing system with blue dye (see our video on the
[Proof of Concept page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27proof-of-concept%27%29%20%7D%7D)
for more). Whilst unidirectional stirring definitely agitated the entire
cross-sectional area of the toroid, there was a noticeable concentration
of dye in one area that was constantly being pushed in the same direction.
Once the stirring system reversed direction, this concentration of dye was
dispersed almost immediately, thereby proving the efficacy of our
bidirectional stirring system.

###### **Measuring Turbidity Through Optical Density**

OD measurement is mandatory for proper turbidostat functionality. Our
thoughts were to use an OD800 LED-phototransistor pair, shining it through
both the bioreactor walls and the culture inside. We opted for OD800 over
OD600 to minimise background radiation interference. Whilst using a laser
initially seemed like a more obvious choice, especially as it would
minimise diffraction possibilities, it quickly became apparent that there
would be issues of self-reflection to contend with if we decided to amend
the design to culture photosynthetic organisms.

The first problem we encountered was difficulty in securing the
LED-transistor pair in place on the bioreactor, so the LED shines directly
on the sensor constantly. Initially we attempted to use male-to-female
jumper wires to connect the LED and phototransistor to the breadboard, but
these were too loose to hold them in place securely. After this we laser
cut a structure that was intended to slot on top of the lid and hold the
LED and phototransistor in place on the sides. This proved much more
promising but the wires of the LED and phototransistor both proved too
short for the pairing to be placed at the correct height from the bottom.
Hence, we soldered wires to increase their lengths which also allowed us
to bend them at angles to each other that were much more suitable to
attaining valid readings.

A second problem we ran into was how short the initial reading range would
be. With the addition of potentiometers we were able to modulate LED
intensity and phototransistor sensitivity, allowing us a greater range of
valid settings to experiment with in our quest for a valid OD range. The
best setup for a valid range is 34.4mA through the LED, and 6.4mA through
the phototransistor.

###### **Controlling Temperature**

Our first attempts at controlling temperature via the Kanthal wire heating
coil left a lot of room for improvement. The temperature would fluctuate a
lot, would only reach up to approximately 31°C but did not reach stability
around 37°C. This is a critical function of our bioreactor, as 37°C is the
optimal temperature for bacterial growth for *V. natriegens*.

Our first attempt at solving this problem was changing the value of the
target temperature in an effort to compensate for temperature
fluctuations. However, simply lifting the benchmark did not prove
adequate. So instead, we used a PID controller to alter heating power
depending on the difference in temperature between the targeted and
current temperature. This has given us much greater control over the
heating coil output.

Another problem we encountered was the motor not turning itself off after
reaching 37°C. We had the heating function in the code controlled by an
if/else loop, so it T>37°C then the motor was supposed to turn itself off.
Our first attempt to fix this utilised a while loop, with the hopes that
it will keep looping through the code and the conditional statement.
However, it turned out that as it looped, the motor would be turned on
again. Our second attempt modified the if/else loop correctly, giving us
greater control. If T<37°C then the motor is on and if T>37°C then the
motor is off.

Finally, we decided to start thinking outside the box, so to speak.
Instead of achieving the desired temperature solely through the heating
coil, we should be focusing more on conserving existing heat output. So,
we fashioned a simple perspex box to act as an insulative cover (10mm
thick walls, and internal dimensions of L160mm × W160mm × H120mm. Perspex
has a thermal conductivity of 0.17 – 0.19 W m/K, which is very low, and
this proved to be sufficient in maintaining optimal temperature ranges for
extended durations.

{% endblock %}