iGEM 2024 — Team khanlabschool

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Our lab notebooks are in five parts as PDFs.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5128/notebook.png)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}To bring our vision to reality we followed the design, build, test, learn engineering cycle and documented the steps we took along the way.{% endblock %}
{% block page\_content %}

Engineering Success

## Overview

### Deciding on a Topic

Our process began with a broad brainstorming session involving the
entire team,
focusing on problems we could address through genetic engineering. We explored various
ideas, such as plant
methane metabolism (enhanced methane removal via plant genetic editing), airborne toxin lung
filtration
(using genetically modified microorganisms to detect and neutralize harmful airborne toxins
before they
enter the respiratory system), plastic decomposition, lead poisoning therapeutics, and fungi
soil resilience
increase (using bioengineered fungi to increase soil resilience).

During our brainstorming session, we heavily researched these ideas
and discussed
each one based on its potential usefulness, impact, and uniqueness. We wanted to choose a
project that would
make a significant difference while also standing out in its approach.

Our results on each topic were as follows:

Plastic Decomposition

* Has been done extensively in the past.
* Our approach would likely involve editing algae
  or E. coli to decompose
  plastic.
* Many plastics already have decomposition methods,
  so we’d
  need to choose a unique target. Initially, we considered PVC, but it proved too toxic to
  work
  with.

Removing Lead from Lungs

* A unique idea with significant potential
  impact.
* However, experimentation would be challenging due
  to the
  complexity of working with human biology.
* It would be difficult to create a solution more
  effective and
  cheaper than EDTA.

Airborne Toxin Filtration (Nitrous Oxide)

* This was an incredibly useful idea, given that
  nitrous oxide is a
  potent greenhouse gas and a significant contributor to air pollution.
* We planned to edit E.
  coli to remove nitrous oxide from the air, which would be
  valuable in areas with high
  industrial emissions.
* However, we found that an iGEM team had already
  attempted
  something similar, limiting the novelty of our approach.

Methane Removal

* Methane is another potent greenhouse gas that
  significantly
  impacts global warming, making its removal from the atmosphere highly beneficial.
* We aimed to edit plants to absorb methane from
  the air, starting
  with E. coli as a proof of
  concept.
* The use of the methane monooxygenase enzyme was
  especially
  promising because, at the time, no one had yet edited E.
  coli with this enzyme.

We ultimately narrowed down our focus to removing greenhouse gasses
from the
environment, but we found it challenging to choose between Airborne Toxin Filtration
(targeting nitrous
oxide) and Methane Removal. Both ideas had significant potential for reducing harmful
emissions and
addressing climate change. For about a month, we invested a considerable amount of time
researching both
approaches.

### Change of Plans

With methane removal, our initial plan was to  genetically
modify an ornamental
tobacco plant (commonly used in genetic edits) to absorb methane. This plant could be placed
near oil
factories and farms to remove methane from the air. Since plants naturally take in large
amounts of air,
this approach seemed more efficient than using regular methanotrophic bacteria. We planned
to start by
editing E. coli and later move on to
the
plant.

However, in December, [a
study](https://www.google.com/url?q=https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11116448/&sa=D&source=editors&ust=1727755367259203&usg=AOvVaw3b-zx4kuWXfnb2RyCF7-MA) came out performing the same experiment. The
scientists also
attempted inserting methane monooxygenase into e coli! It was exciting to see scientists
with the same idea
as us perform the experiment, but it meant we were back to the drawing board.

We revisited our earlier idea involving nitrous oxide. We considered
the possibility
of genetically editing a plant to remove both methane and nitrous oxide, as they are often
found in the same
environments. However, we soon realized that executing both genetic edits would be too
time-consuming,
especially since it was our first time as an iGEM team. Ultimately, because our idea for
airborne toxin
filtration with nitrous oxide was to genetically modify plants to turn nitrous oxide into
nitrogen via
nitrous oxide reductase, which was not a novel idea in the scientific community, we shifted
our focus to
methane completely.

As we researched more on methane, we identified landfills as the
third-largest source
of methane emissions. Methanotrophs, or methane-eating bacteria, would not be effective in
this environment
due to their limited ability to survive and thrive in landfill conditions. This led us to
consider Bacillus
subtilis, a bacterium commonly found in soil and far more abundant than typical
methanotrophs.

## Design 1

Following the steps a group from the University of Korea did, we
replicated the work
done in [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11116448/](https://www.google.com/url?q=https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11116448/&sa=D&source=editors&ust=1727755367260037&usg=AOvVaw0GVOs1TU-eM2pnUp-8099T) to build the protein sequence for the mini-sMMO. From there, we used
IDT’s codon
optimizer to optimize for B. subtilis, resulting in our final DNA sequences for the
mini-sMMO alpha and beta
subunits.

[This
document](https://static.igem.wiki/teams/5128/minismmosummary.pdf) contains a summary of the mini-sMMO article,
the protein
sequence for the mini-sMMO, and the DNA sequence for the mini-sMMO.

We designed a construct using that sequence with Leah, a graduate
student from the
University of Wisconsin's, help.

![](https://static.igem.wiki/teams/5128/engineering/image6-min.png)

![](https://static.igem.wiki/teams/5128/engineering/image8-min.png)

Our final linear DNA is composed of 4 main fragments: a pDR110
segment, the mini-sMMO
alpha subunit, the mini-sMMO beta subunit, and another pDR110 segment. (see first diagram).
The front and
the back of our linear DNA product is amyE back and amyE front, which are the homologous
parts of our DNA
that allow it to replace the amylase gene of B. subtilis’s chromosomal DNA.

## Build 1

We PCRed fragment one and fragment four from PDR110, and ordered
fragment two and
fragment three from IDT. There were challenges in that process, fragment two failed
IDT’s quality
checks, and so they had to send it to us in two parts. To address this we ran
overlap extension PCR to combine the two parts IDT gave us, and
then tried to
combine all our parts using Gibson assembly.

![](https://static.igem.wiki/teams/5128/engineering/image4-min.jpg)

(Left to right): ladder, fragment 1 (2.6k), blank, ladder, fragment 4
(2.1k)

Using a NEB 1 kb+ ladder.

## Test 1

We decided to split our testing into four groups. Two sets, each with
a different
glycerol stock were created. Within each set we transformed PCRd and non PCRd versions of
the Gibson
Assembly. Unfortunately we got zero transformants. To test further, ran a gel on our PCRd
gibson, which led
us to a realization: Our PCR results were 3000 bp shorter than expected.

![](https://static.igem.wiki/teams/5128/engineering/image7-min.png)

[left to right] PCRd gibson, NEB 1 kb+ ladder

## Learn 1

We started considering what went wrong, and then we realized the
4.6kb PCR product
lined up with the size of just fragment one and fragment four, without our insert. So we
tried to consider
what could have produced a result of that size.

We eventually realized that the primers we were using would amplify
any DNA starting
with amyE back and ending with amyE front, including our plasmid (PDR110). Our plasmid likely entered our
Gibson from the unpurified fragment one and
fragment four.

![](https://static.igem.wiki/teams/5128/engineering/image3-min.png)

## Design 2

We started to rethink our lab protocol, with help from Leah. The
major changes we
made were:

* Start with PCR purified DNA.
* Not PCRing the gibson to avoid amplifying
  portions of
  PDR110.
* Circular DNA doesn’t transform well, so
  plasmid leftovers
  should be fine.
* Use simpler competent cell prep

## Build 2

We returned to the lab again, re-running PCR on all of our fragments
(there are four
official ones, but fragment 2 was shipped to us in two parts by IDT giving us a total of
five DNA sequences
to amplify. We ran three reactions of each, ran a gel on ten ul of each, and pooled the
remainder for PCR
purification. We then ran a five way Gibson assembly, and transformed the results into our
newly prepared
competent cells.

![](https://static.igem.wiki/teams/5128/engineering/image1-min.png)

[from right to left] ladder, frag 1, frag 2a, frag 2b, frag3, frag 4,
frag 1, frag
2a, frag 2b, frag 4, blank, frag 1, frag 2a, frag 2b, frag 4.

![](https://static.igem.wiki/teams/5128/engineering/image1-min.png)

Zoomed in version of above only including [from right to left]
ladder, frag 1, frag
2a, frag 2b, frag3, frag 4

## Test 2

This time we had transformants! However when we let the plates grow
longer we also
ended up with colonies on our negative control. To confirm that the colonies were actually
successful
transformants, we plated them on new
plates with starch
and added iodine. If transformation was successful the bacteria would not be able to consume
tha starch
(amylose) due to a knocked out amylase gene.

Iodine reacts with starch to turn purple, and otherwise remains
yellow, so successful
transformants should have purple covers, whereas unsuccessful ones should have yellow/orange
hallows.

![](https://static.igem.wiki/teams/5128/engineering/image5-min.png)

![](https://static.igem.wiki/teams/5128/engineering/image2-min.png)

## Learn 2

We learned the hard way that spectinomycin, our antibiotic, actually
degrades
significantly over the course of a week at 37 C, resulting in contamination. Our starch test
results are not
conclusive, so we would like to run further tests to confirm or deny the production of sMMO.
We also hope to
confirm that our Gibson reaction actually worked by sending it in for sequencing.

In the end, by following the engineering cycle we were able to go from a vision in our heads of B. subtilis absorbing methane to live transformants of genetically engineered B. subtilis in the lab. While we do still have additional lab work to do, we believe this along with our work on hardware demonstrates engineering success.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}The research, experiments, and protocols we used in our iGEM project.{% endblock %}
{% block page\_content %}

## Dilution standards:

|  |  |
| --- | --- |
| Name | Concentration |
| Gene block | 10 ng/ul |
| Primers | 25 nmol/ul |

##

## SMMO Fragment PCR protocol:

1. [https://www.neb.com/en-us/protocols/2012/08/30/protocol-for-q5-hot-start-high-fidelity-2x-master-mix-m0494](https://www.google.com/url?q=https://www.neb.com/en-us/protocols/2012/08/30/protocol-for-q5-hot-start-high-fidelity-2x-master-mix-m0494&sa=D&source=editors&ust=1727752276681995&usg=AOvVaw0bLq9BeNAcASJBCqxbTrJH)
2. To a pcr tube add 2 ul of forward primer a and reverse
   primer a (10 uM), and
   25 ul of Q5 Hot Start High-Fidelity 2X Master
   Mix.
3. Add 10 ul of 10 ng/ul template DNA fragment
   a, and 11 ul of milliQ or
   dH20 to the tube.
4. Repeat steps two and three in a second tube
   for fragment b and its
   associated primers.
5. Place both tubes in a thermocycler and run
   at the temperatures
   specified below.
6. To confirm successful amplification, take 10 ul from
   each tube into a separate
   tube and add 2 ul of Gel Loading Dye, Purple (6X), no
   SDS to
   each tube.
7. In the first well, place 10 ul of 1 kb Plus DNA Ladder
   for Safe Stains, in the subsequent to wells, place the
   15 ul of prepared solution from
   the tubes with loading dye.
8. Run the gel and confirm that fragments a and
   b have visible lines at
   ~2100 and ~1100 bp respectively.
9. Confirm fragment concentrations with
   nanodrop.

|  |  |  |
| --- | --- | --- |
| STEP | TEMP | TIME |
| Initial Denaturation | 98°C | 30 seconds |
| 30 Cycles | 98°C  45-68°C  72°C | 15-30 seconds  15-60 seconds  60(s) for fragment A, 30(s) for fragment B. - 30 second per kb |
| Final Extension | 72°C | 5 minutes |

## Plasmid fragment PCR protocol:

1. Repeat the Smmo fragment pcr protocol with
   new forward and reverse
   primers. You will need two sets of forward and reverse primers, one for the
   front end and one for the back end
   of the amye gene.
2. The fragments produced will be shorter, so
   the extension time in the
   pcr cycle can be reduced to one minute.

## PCR Fragment purification:

1. [https://zymoresearch.wufoo.com/forms/w1p5d1kg0a6kw05/](https://www.google.com/url?q=https://zymoresearch.wufoo.com/forms/w1p5d1kg0a6kw05/&sa=D&source=editors&ust=1727752276688067&usg=AOvVaw3hmmRUwexSdA9Zt8K4t0ad)
2. [https://files.zymoresearch.com/protocols/\_d4003t\_d4003\_d4004\_d4013\_d4014\_dna\_clean\_concentrator\_-5.pdf](https://www.google.com/url?q=https://files.zymoresearch.com/protocols/_d4003t_d4003_d4004_d4013_d4014_dna_clean_concentrator_-5.pdf&sa=D&source=editors&ust=1727752276688610&usg=AOvVaw0x3a_bhD7kh9LOu1t0IdJM)
3. Run all fragments that have been multiplied
   through pcr purification,
   measure their final concentrations with the nanodrop

## DNA assembly protocol:

1. [https://www.neb.com/en-us/protocols/2014/11/26/nebuilder-hifi-dna-assembly-reaction-protocol](https://www.google.com/url?q=https://www.neb.com/en-us/protocols/2014/11/26/nebuilder-hifi-dna-assembly-reaction-protocol&sa=D&source=editors&ust=1727752276689155&usg=AOvVaw0UaHqDvQ02NheXYksQ3p1_)
2. Add a 10 ul of NEBuilder HiFi DNA Assembly
   Master Mix to a tube along
   with ~0.10 pico moles of each DNA fragment.
3. Add dH20 until the reaction volume is 20
   ul.
4. Heat to 50 C for one hour.
5. Run a gel with 10 ul of the mixture to
   confirm the ligation of the
   fragments.

## Competent cell prep (complex):

1. [https://static.igem.org/mediawiki/2020/7/71/T--Brno\_Czech\_Republic--prot-test-com.pdf](https://www.google.com/url?q=https://static.igem.org/mediawiki/2020/7/71/T--Brno_Czech_Republic--prot-test-com.pdf&sa=D&source=editors&ust=1727752276689848&usg=AOvVaw0iz3YI1JQDHMFuGtTWGzpj)

## Antibiotic IPTG plate prep:

Make IPTG stock (0.100mM - serves as 1000x for
plates):

1. [https://goldbio.com/product/1379/iptg](https://www.google.com/url?q=https://goldbio.com/product/1379/iptg&sa=D&source=editors&ust=1727752276690336&usg=AOvVaw1EOYHGdQUghoijFmcj7Seo)
2. Weigh 0.238 g of IPTG
3. Add 10 ml sterile H2O. Dissolve
   completely.
4. Prewet a 0.22 µm syringe filter by
   drawing through 5-10 ml of
   sterile H2O and discard water.
5. Sterilize IPTG Stock through the prepared [0.22
   µm syringe filter](https://www.google.com/url?q=https://www.amazon.com/Membrane-Solutions-Syringe-Filter-Sterile/dp/B09NNBXNR3&sa=D&source=editors&ust=1727752276690825&usg=AOvVaw21jzCMDnfqzmG-dL5hVmQ8).

Make Spectinomycin stock:

1. Add 100 mg of spectinomycin to 1 ml of
   dh2O.
2. Vortex, then run to a 0.22 um filter to
   sterilize.

Make plates:

1. [https://static.igem.org/mediawiki/2014/5/59/LMU\_Munich14\_Media.pdf](https://www.google.com/url?q=https://static.igem.org/mediawiki/2014/5/59/LMU_Munich14_Media.pdf&sa=D&source=editors&ust=1727752276691825&usg=AOvVaw3y550Q25Xchfa75EAqp0mG)
2. [https://agscientific.com/blog/iptg-facts-protocols.html](https://www.google.com/url?q=https://agscientific.com/blog/iptg-facts-protocols.html&sa=D&source=editors&ust=1727752276692176&usg=AOvVaw0rgGsd4jxMZ93ngJcjqQBG)
3. [https://www.addgene.org/protocols/pouring-lb-agar-plates/](https://www.google.com/url?q=https://www.addgene.org/protocols/pouring-lb-agar-plates/&sa=D&source=editors&ust=1727752276692486&usg=AOvVaw1PwvkJW7DHPZwWsaSTGdNd)
4. Reheat 40 ml of premixed lb agar in a water
   bath and add 40 ul of IPTG
   stock (0.100mM) and 40 ul of Spectinomycin stock once the temp is ~ 60 C.
5. Stir to combine, then pour four
   plates.

## Transformation protocol:

1. [https://static.igem.wiki/teams/4417/wiki/protocols/bacillus-subtilis-transformation-protocol-created-by-ucl-igem-2022.pdf](https://www.google.com/url?q=https://static.igem.wiki/teams/4417/wiki/protocols/bacillus-subtilis-transformation-protocol-created-by-ucl-igem-2022.pdf&sa=D&source=editors&ust=1727752276693217&usg=AOvVaw1S-zJrakJ7-P_BWrj8xkOV)
2. Take a 500 ul sample of competent cells, and
   add 500 ng of ligated DNA
   to it in an sterile 1.5 ml tube.
3. Incubate at 37 C/250 rpm for 1 hour.
4. Spread mixture onto prepared spectinomycin
   plates, incubate at 37 C
   for 1-2 days.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}Below are the results that we achieved.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5128/results/image4-min.png)

## Expression of mini-sMMO in B. subtilis

Our DNA construct (part BBa\_K5128100) is based on the plasmid pDR110 and
mini-sMMO gene
fragments. The amyE front and amyE back sections enable mini-sMMO to be inserted into the
middle of the
B. subtilis amylase (amyE) gene via
homologous
recombination. The spectinomycin resistance gene specR enables us to screen for transformed
colonies. Since
methanol, the product of mini-sMMO acting on methane, is toxic to B. subtilis, we also
included the lacI
operon, so that mini-sMMO will be expressed only when IPTG is added.

![](https://static.igem.wiki/teams/5128/results/image6-min.png)

We ordered mini-SMMO gene fragments from [IDT](https://www.google.com/url?q=https://www.idtdna.com/&sa=D&source=editors&ust=1727757222347261&usg=AOvVaw3T3-eSbNyqdr_r8_ySnkZU), but IDT was unable to synthesize the gene fragment for the alpha subunit.
Subsequently, we
re-designed the alpha fragment and split it into two parts, which we then re-ordered. Using
overlap-extension PCR, we stitched together these two parts into the alpha fragment.

![](https://static.igem.wiki/teams/5128/results/image8-min.jpg "PXL_20240907_204603571.jpg")

The left lane is the [New
England Biolabs 1 kb Plus DNA Ladder](https://www.google.com/url?q=https://www.neb.com/en-us/products/n3200-1-kb-plus-dna-ladder&sa=D&source=editors&ust=1727757222347825&usg=AOvVaw11gayX40-FbnsjEyd4aFDr), and the right lane
is the
overlap-extension PCR product. The bright band in the right lane is consistent with 2600 bp,
the length of
our alpha fragment.

We assembled our four fragments (amyE back, alpha fragment, beta
fragment, amyE
front) using [NEBuilder
HiFi DNA Assembly](https://www.google.com/url?q=https://www.neb.com/en-us/applications/cloning-and-synthetic-biology/dna-assembly-and-cloning/nebuilder-hifi-dna-assembly&sa=D&source=editors&ust=1727757222348256&usg=AOvVaw0M6OC1RdSMJsgfKE7pTrIt) (similar to Gibson assembly). This
was then
transformed into B. subtilis strain 168
and streaked on
LB/agar/spec plates to grow at 37°C overnight. This first transformation attempt was
unsuccessful, as we
ended up with no colonies on our plates.

For our second transformation attempt, we skipped the
overlap-extension PCR and used
the NEBuilder HIFi Assembly to directly assemble the five fragments (amyE back, alpha\_1
fragment, alpha\_2
fragment, beta fragment, amyE front). We also ran our PCR in triplicate to ensure that we
had plenty of
starting material to work with, and we added a quality control step by verifying our PCR
products on a
gel.

![](https://static.igem.wiki/teams/5128/results/image2-min.png)

From right to left: ladder ([New
England Biolabs 1 kb Plus DNA Ladder](https://www.google.com/url?q=https://www.neb.com/en-us/products/n3200-1-kb-plus-dna-ladder&sa=D&source=editors&ust=1727757222348992&usg=AOvVaw3kzVcwAKhFAUeX6PlTGFrc)), amyE back, apha\_1,
alpha\_2, beta,
amyE front, and then the 5 fragments are repeated in order. Each of the lanes have clear
bands at the right
position.

Following assembly, the product was transformed into B.
subtilis strain 168 and streaked on LB/agar/spec plates to grow
at 37°C
overnight. After the first night, we saw three colonies. After the second night, we saw many
more colonies,
including on the negative control plates, so we decided to move ahead with just the three
colonies from the
first night incubation. We re-streaked each of the three colonies on LB/agar/spec plates and
on LB/starch
plates.

Following incubation at 37°C overnight, we saw growth on all the
plates. On the
LB/starch plates, we performed the iodine-starch test. Starch and iodine combine to make a
dark purplish
color, and a yellowish halo around a colony indicates a deficit of starch around the colony,
suggesting that
the amylase gene (amyE) is present. Conversely, a lack of a halo suggests that amyE is
absent and that the
colony has been successfully transformed.

![](https://static.igem.wiki/teams/5128/results/image5-min.png)

AmyE+ indicates that the amylase gene is still present, whereas,
AmyE- indicates
that the amylase gene is not present, suggesting a successful transformation.

We interpret the starch test as showing that one of the colonies is amyE
negative. Going
forward, we plan to do further experiments to check if that colony has truly been
successfully
transformed.

## Thermocycler

Our team developed a Peltier-module-based PCR machine that can be built for half
the price of
the cheapest available commercial offering. The design and code is shared on [GitHub](https://www.google.com/url?q=https://github.com/Ansh-123/Peltier-based-PCR&sa=D&source=editors&ust=1727757222350367&usg=AOvVaw1X23HRozzamFXc9f2XhlMa). Despite the shortcomings of our design, such as the lack of a heated lid and
its unwieldiness,
we were able to perform a successful test run during our “Get Ready for Synthetic
Biology” class
that we taught to middle school students. In the future, we hope to update the PCB design to
significantly
reduce the amount of wiring and work to select more temperature resistant Peltier
modules.

![](https://static.igem.wiki/teams/5128/results/image7-min.png)

An image of the thermocycler

![](https://static.igem.wiki/teams/5128/results/image1-min.png)

A gel that we ran on the PCR product from our thermocycler

## Microcentrifuge

We successfully developed a microcentrifuge rotor designed to fit on top of a
standard 1000kv
drone motor. The design is shared on [Thingiverse](https://www.google.com/url?q=https://www.thingiverse.com/thing:6729835&sa=D&source=editors&ust=1727757222351371&usg=AOvVaw2YzsTtL9f1tT--cOPL5DLc). This brought the cost of the microcentrifuge down to $35, including the cost of
3D printing,
which is less than half the price of the cheapest online model. By filming a slow-motion
video of the
centrifuge in action, we estimated the rpm of the microcentrifuge and calculated an RCF of
5000g. This
value, while not anywhere close to more expensive lab grade centrifuges, was quite close to
the cheapest
online model that we found. In the future, we hope to design an enclosure for the centrifuge
to increase
safety.

![](https://static.igem.wiki/teams/5128/results/image3-min.png)

An image of the microcentrifuge

{% endblock %}