iGEM 2025 — Team nu-boston

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{% block title %}Notebook{% endblock %}
{% block lead %}Wet Lab{% endblock %}
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# Lab Notebook

##### Navigation

[May 2025](#may2025)
[June 2025](#june2025)
[July 2025](#july2025)
[August 2025](#august2025)
[September 2025](#september2025)

### May

1

#### May 12 - May 18

Ankee

This week marked our first official week in the lab for the NU
iGEM Team. We planned to begin growing bacterial cultures of
Luke-2, RFP, pET28a\_CBM\_CohIII, and pET28a\_Xyl\_Doc. However,
since only the RFP plasmid had arrived, we proceeded with that
while waiting for the remaining constructs to be delivered. To
support upcoming experiments, we prepared fresh kanamycin
plates and aliquoted several antibiotics, including kanamycin,
ampicillin, and chloramphenicol for future use. We also made
bacterial growth medium and LB agar to establish baseline
materials for the team. Finally, we performed plasmid
purification on the RFP plasmid. The nanodrop measurement
showed a concentration of 123.65 ng/µL, which gave us a great
starting point for subsequent experiments.

2

#### May 19 - May 25

Amy
Katya

This week, we purified the RFP plasmid. Initially, plasmid DNA
was amplified via polymerase chain reaction (PCR) using the Q5
2× Master Mix to generate multiple copies of the RFP sequence.
The PCR products were subsequently purified using the Monarch
PCR purification kit, followed by agarose gel electrophoresis
to confirm the presence of the RFP plasmid. The purified
products were then quantified using a spectrophotometer. In
addition, the pET28a plasmid was transformed into E. coli
BL21DE3 cells.

3

#### May 26 - June 1

Amy
Katya

We repeated the transformation of the pET28a plasmid into E.
coli DH5a cells. Following transformation, the cultures were
plated using either 100 µL, 200 µL, or 200 µL of resuspended
culture. After overnight incubation, individual colonies from
each plate were selected and inoculated into separate cultures
for further growth.

### June

4

#### June 2 - June 8

Amy
Katya

We initiated cultures of pET28a-RFP in BL21(DE3) cells by
picking clones from a transformation plate. In preparation for
creating an RFP iGEM logo, we poured media into the plate
design and incorporated LB agar containing IPTG. The
pET28a-RFP plasmid was also purified. The logo plates were
then streaked with the RFP culture, and fluorescence imaging
was performed to test RFP expression and logo visualization.

5

#### June 9 - June 15

Amy
Katya

We transformed Puc19 as a control and plated the cells on
ampicillin plates. In addition, we prepared another agar plate
with the iGEM logo, streaked it with RFP, and imaged the
resulting plates. We also conducted preliminary burn tests
using cotton coated with different concentrations of DNA.
Finally, overnight cultures were prepared for use in the
following week.

6

#### June 16 - June 22

Amy
Katya

We transformed pET28a\_CBM\_CoHIII and pET28a\_DocIII\_RFP into
BL21(DE3) cells. Following transformation, 100 µL, 200 µL, or
200 µL of resuspended cultures were plated onto kanamycin
plates and incubated overnight. Colonies from each plate were
then selected to initiate 50 mL cultures. Also, we performed a
back-dilution of Puc19.

To assess protein expression, we generated a protein
production growth curve by measuring OD values of the CBM and
RFP cultures every 30 minutes over a six-hour period. Protein
binding activity was also evaluated by submerging cotton
samples into supernatants containing either CBM-CoHIII,
RFP-DocIII, a mixture of both, or Triton X-100 alone. We also
ran some flame-resistance tests of cotton coated with either
protein or DNA, measuring how long it took for the cotton to
fully burn.

7

#### June 23 - June 29

Amy
Katya

This week, we started cultures of several different
carbohydrate-binding modules. We transfected 50 mL cultures
with either Luke-2 (with ampicillin selection) or
pET28a-CohII-CBM-HIS-YBBR (with kanamycin selection) and
incubated them at 37 °C. We also picked colonies from the
pET\_CBM\_CoHIII and pET\_RFP\_DocIII plates to start three 50 mL
cultures of each construct (six total). After sufficient
growth, we prepared glycerol stocks of each construct and
stored them at -80°C. Additionally, we performed a PCR of
pET28a, confirmed the product via gel electrophoresis, and
completed a PCR cleanup to generate enough plasmid for next
week's Gibson assembly.

### July

8

#### June 30 - July 6

Amy
Katya

We constructed five different plasmids using Gibson assembly
with pET28a as the backbone and Alpha-S-Casein,
Beta-Lactoglobulin, PurA, Soybean Glutenin, or Streptavidin as
inserts. The assembled plasmids were then transformed into
competent BL21 cells and plated for incubation. In parallel,
we initiated additional 50 mL cultures of Luke-2 and
CBM-CohIII-YBBR-HIS, which we incubated overnight and then did
a plasmid prep. Following incubation, the transformation
plates showed limited growth. So, we picked clones and
performed colony PCR only on the three constructs that
displayed some growth (Alpha-S-Casein, Soybean Glutenin, and
Streptavidin). We also repeated the transformations for all
the constructs, this time using 10 µL of culture instead of
the 2 µL used previously.

9

#### July 7 - July 13

Amy
Katya

We prepared more Puc19 cultures. We also re-ran a protein gel
of CBM-CoHIII and RFP-DocIII, and started more cultures of
pET\_RFP\_DocIII and pET\_CBM\_CohIII. Then, we ran a PCR on
Pet28a. Additionally, we measured a protein growth curve of
the RFP-DocIII cultures and CBM-CoHIII cultures, both induced
and non-induced (four cultures total).

10

#### July 14 - July 20

Kristina
Pablo

This week the team focused on maintaining and preparing
bacterial cultures. Early in the week, 5 mL of the existing
culture was transferred into a labeled 500 mL flask containing
45 mL of LB media and 50 µL of kanamycin to sustain selective
growth. Cell density was monitored by measuring OD600 on the
NanoDrop. Throughout the process, detailed notes were recorded
on setup and handling procedures to ensure reproducibility and
streamline future culturing work.

11

#### July 21 - July 27

Kristina
Pablo

Protein samples were analyzed by SDS-PAGE using pre-cast
polyacrylamide gels run at 180V for 1.5 hours, followed by
Coomassie Blue staining and gel imaging. Colony PCR was
performed using OneTaq MasterMix with construct-specific
thermal cycling conditions (94°C denaturation, 49°C annealing,
68°C extension) to screen transformants containing
AlphaSCasein, Soybean Glutenin, BetaLactoglobulin,
Streptavidin, and PurA constructs. PCR products were analyzed
on agarose gels at 140V for 30-45 minutes. Plasmid DNA was
isolated from overnight cultures and quantified by nanodrop
spectrophotometry. Bacterial transformations were conducted
using standard heat shock protocol (30 min on ice, 42°C for 10
sec, 5 min on ice) followed by Super Optimal broth with
Catabolite repression recovery at 37°C for 1 hour and plating
on selective media.

### August

12

#### July 28 - August 3

Kristina

Alpha-S-Casein constructs were submitted for sequencing and
Gibson assemblies were performed using Q5 2x Master Mix with
optimized ratios, followed by overnight DpnI digestion at
37°C. Transformations utilized NEB 5-alpha competent cells
with heat shock protocol, and protein expression analysis was
conducted using BL21(DE3) strains with IPTG induction at
mid-log phase. Growth curves were monitored by OD600
measurements and glycerol stocks were prepared for long-term
storage. Colony PCR verification employed OneTaq MasterMix
with products analyzed on 1% agarose gels.

13

#### August 4 - August 10

Kristina

Protein expression analysis was conducted using SDS-PAGE with
cell pellets resuspended in PBS and prepared with Laemmli
buffer at 95°C. Gels were run at 100V for 10 minutes followed
by 150V for 50 minutes, then stained with Coomassie Blue.
Plasmid preparations were quantified by nanodrop
spectrophotometry and submitted for sequencing with
appropriate sample protection. Transformations utilized
BL21(DE3) competent cells with heat shock protocol (30 min
ice, 42°C for 45 sec, 2 min ice) followed by SOC recovery and
plating on Kanamycin-selective media. Growth curves employed
OD600 monitoring with IPTG induction at appropriate cell
density, tracking both induced and non-induced conditions for
comparative analysis. Alternative incubation temperatures
(4°C, -20°C) were successfully implemented when standard ice
incubation was unavailable.

14

#### August 11 - August 17

Kristina

This week we analyzed protein via SDS-PAGE of 3-hour samples
from five constructs (ASC, BLG, SBG, PurA, Strep) under
induced and non-induced conditions, and transformation of
Trans7 and Trans5 DNA constructs into BL21DE3 cells. Key
issues included improper SOC storage at room temperature
instead of 4°C and insufficient gel wells requiring additional
runs for Strep and PurA spindown samples. Transformation
plates were prepared with kanamycin selection and gel imaging
was completed successfully.

15

#### August 18 - August 24

Kristina

This week encompassed multiple molecular biology techniques
including transformation verification, protein analysis, PCR
amplification, and Gibson assembly preparation. Transformation
plates from Week 14 showed successful colony growth and were
moved to 4°C storage, followed by bacterial stock preparation
using glycerol. ASC protein samples from August 9th were
analyzed via SDS-PAGE, focusing on 3-hour time points under
induced and non-induced conditions. PCR amplification of
pET28a plasmid was performed using iGEM-004/006 primer pairs,
followed by DpnI digest for template removal. The week
concluded with preparation for Gibson assembly, including
gBlock reconstitution and plasmid quantification.

16

#### August 25 - August 31

Pablo

This week we focused on protein characterization and cloning
procedures through SDS-PAGE analysis and PCR amplification.
Seven ASC protein samples from August 9th (Pre, 1 Hr I/NI, 2
HR I/NI, and 3 HR I/NI) were analyzed via gel electrophoresis
after preparation with Laemmli buffer containing
β-mercaptoethanol, with samples boiled at 95°C before running
at 150V for one hour. PCR amplification of pET28a plasmid was
performed using iGEM-005/006 primer pairs with Q5 2x Master
Mix, running 28 cycles at 62°C annealing temperature.
Following PCR completion, DpnI was added for overnight
digestion at 37°C to remove template DNA. The week concluded
with gel staining overnight and storage of both protein
samples at -20°C and PCR products in the thermocycler for
subsequent processing.

### September

17

#### September 1 - September 7

Pablo

This week we focused on troubleshooting protein expression
issues through colony PCR verification and protein gel
analysis. Due to inconsistent protein gel results, the team
initiated diagnostic procedures by streaking five bacterial
strains (iGEMS\_024 through iGEMS\_028) onto Kanamycin-50
plates. Colony PCR was performed using iGEM25-121 and
iGEM25-151 primers with OneTaq MasterMix, running
strain-specific cycling conditions for ASC, β-lactoglobulin,
streptavidin, soybean glutenin, and PurA. DNA gel analysis
confirmed plasmid presence in all samples. Additionally, two
gBlocks (Cipa\_RFP and CbpA\_RFP) were reconstituted to
approximately 11-12 ng/μL for Gibson assembly preparation with
pET28a backbone. The week concluded with SDS-PAGE analysis of
protein samples from various time points (Pre, 1HR I/NI, 2HR
I/NI, 3HR I/NI), comparing both regular and centrifuged
samples to assess protein expression levels. An iGEM\_RFP
culture was also initiated for future experiments.

18

#### September 8 - September 14

Pablo

This week we focused on Gibson assembly optimization and
temperature-dependent protein expression studies. Gibson
assembly was performed using pET28a backbone (13.7 ng/μL) and
CbpA\_RFP gBlock (11.93 ng/μL) with NEBuilder Master Mix,
incubated at 50°C for one hour. Following cleanup with NEB PCR
purification kit, the assembly product was transformed into
DH5α competent cells and plated on Kanamycin-50 plates.
Protein expression optimization involved testing iGEMS\_024
cultures under varying conditions: two temperatures (32°C and
37°C) and three induction states (1mM IPTG, 250μM IPTG, and
non-induced). Growth monitoring over six hours revealed
differential expression patterns, with 37°C cultures showing
higher OD600 values (reaching 2.74 for non-induced) compared
to 32°C conditions (maximum 2.60). Hourly samples were
collected, centrifuged, and stored at -20°C for subsequent
protein analysis. The week's work established temperature and
IPTG concentration parameters for optimizing protein
expression in the system.

19

#### September 15 - September 21

Pablo

This week we continued optimizing our Gibson assembly and
comprehensive protein expression analysis under varying
conditions. Multiple attempts at Gibson assembly were
performed using pET28a backbone and RFP\_CbpA gBlock, followed
by transformation into DH5α cells. Protein expression studies
compared iGEMS\_024 and iGEMS\_025 strains at two temperatures
(32°C and 37°C) with different IPTG concentrations (250μM and
1mM). SDS-PAGE analysis of 2-hour and 3-hour timepoints
revealed variable expression patterns across conditions. BCA
assay was performed to quantify protein concentrations using
ethylene glycol-diluted albumin standards, with samples
normalized to OD 10. The week concluded with PCR product
cleanup yielding concentrations of 81.9 ng/μL and 145.7 ng/μL
with good purity ratios. Additionally, PUC19 glycerol stocks
were initiated as backup cultures, and transformation plates
showed colony growth requiring further screening.

20

#### September 22 - September 28

Lavanya

This week we ran a DNA gel, SDS-PAGE gel, transformation into
DH5α cells, and harvesting cells for protein extraction. We
began by running and imaging a gel of both samples of pET28a.
A NEBuilder HiFi reaction was performed using pET28a and Cipa,
and an SDS-PAGE gel was run using samples from August 9th.
These samples included StrepPre, Strep1HR NI, Strep1HR I,
Strep2HR NI, Strep2HR I, Strep3HR NI, Strep3HR I, Strep Pre
Sup, Strep1HR NI Sup, Strep1HR I Sup, Strep2HR NI Sup,
Strep2HR I Sup, Strep3HR NI Sup, and Strep3HR I Sup. The gel
was left to stain overnight, and the results of the HiFi
reaction were transformed into DH5α cells. Serial dilutions
were performed during the protocol and plated for overnight
incubation. Overnight cultures of iGEM\_024 and iGEM\_025 were
also created for later use. These cultures were then
back-diluted into fresh LB with kanamycin, and once OD600
measurements reflected growth of 0.22 and 0.19, respectively,
they were induced with IPTG. Both non-induced and induced
cultures were harvested by centrifugation, resuspended in 0.5%
Tween20, and lysed using a NaOH and cellulose solution. They
were then treated with ethylene glycol, spun down, and the
supernatant was collected and stored.

##### Lab Notebook Entry

Close

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{% block title %}Engineering & Results{% endblock %}
{% block lead %}Blueprint for Engineering Excellence: Our DBTL Journey{% endblock %}
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# Engineering & Results

##### Navigation

[DBTL #1](#dbtl1)
[DBTL #2](#dbtl2)
[Burning Testing](#BurningTesting)
[References](#references)

## DBTL #1

---

### Design

The purpose of this project is to confer flame retardancy to plants and
other cellulose substrates. To do this we need to identify natural flame
retardants and identify strong cellulose binding domains.

To start our project we started with the flame retardant proteins. Our
research revealed three main characteristics that a flame retardant
material must have in order to make mechanisms for protein flame
retardants to work. An acid source, a carbon source and a blowing agent
which releases large quantities of gases (1). A material with these
characteristics is called an intumescent material. These act to form an
insulating char which acts to prevent reigninition. It may burn first
but then will not reignite, or will burn far slower (2). Another method
is to release water or non-flammable gases while burning, such as
nitrogen or other inert gases. This will act to either fully extinguish
the flame or again prevent the spread of the fire.

To expand a bit as the protein heats up it begins to degrade. As it
degrades gases such as nitrogen are released from the structure of the
protein and into the surrounding area. This deprives the area of oxygen
and contributes to the formation of a char layer. Proteins are also very
good at forming a char layer due to their polymeric properties. This
char layer protects materials below it and also prevents the fire from
spreading. Flame retardants high in phosphorus and nitrogen have been
shown to be the best performing systems (3).

While many proteins could potentially satisfy these requirements,
certain proteins such as Caseins, Glutens, Soy Proteins and Gelatin have
been identified as being good flame retardants (3) When exposed to heat
Caseins form an expanded carbon structure filled with bubbles which
prevents and limits the spread of the fire (4). Additionally, Caseins
are abundant in glutamines which contain -NH2 groups, which are primed
to produce nitrogen gas to form that inhibitory char (5).

Beyond caseins, Whey proteins are another type of protein that have been
identified as potentially having strong flame retardant properties. This
is because the even distribution of hydrophilic amino acids allows the
proteins to have fantastic water absorbent properties, additionally whey
proteins can form a strong gas barrier (5). Beta-lactoglobin is the
primary component of whey proteins. Finally we found that soy is able to
form an oxygen-limiting char which slows a flame down (6).

During the course of our research we discovered that DNA is also an
incredible flame retardant. As mentioned previously flame retardants
high in phosphorus and nitrogen have been shown to be the most
effective. Additionally DNA contains all the necessary components of an
intumescent system; the phosphate backbone acts as an acid, the
deoxyribose acts as a carbon source and then finally the bases contain a
lot of nitrogen which act as a blowing agent.

While DNA is a strong flame retardant it does not stick to cotton. So,
we will instead use proteins to bind to DNA to attach it to the
cellulose (7). To bind our proteins we settled on PurA, which is a
single stranded binding protein that binds to purine rich sequences (8).
Finally, since it is important for our proteins to bind tightly together
we decided to experiment with the streptavidin and biotin interaction by
purchasing biotinylated nucleotides and linking Streptavidin to a
cellulose binding domain. In the meantime while working on our cellulose
binding domains we will also transform pET28a into DH5a and purify in
large quantities in order to verify the flame retardancy of DNA. Thus
after our research we had settled on five proteins to test the flame
retardant properties:

* Alpha-S-Casein from Bos Tarus
* Beta-LactoGlobin from Bos Tarus
* Soy BeanGlutenin
* PurA from E.coli
* Streptavidin from Streptomyces Avidinii

Now that we have our flame retardant proteins we needed a way to attach
our proteins to cellulose. We settled on the cellulosome complex. This
consists of a cellulose binding domain and then a dockerin and a
cohesion. While naturally for the digestion of cellulose they can be
used to attach to cellulose (9). While there are multiple different
types of cellulose binding domains, we settled on the cellulose binding
domain from the bacteria Clostridium Thermocellum due to its low Ka
value (10).

We decided to start with the cellulose binding domain from Clostridium
Thermocellum and decided to create a fusion protein of CbpA conjugated
to CohIII. Additionally we made an RFP-DocIII in order to quantitatively
assess the binding capability of the different cellulose binding
domains.

### Build

In order to make our constructs we pulled sequences from FASTA and then
codon optimized for production in E.coli. At the end of our design
stages we had settled on five possible cellulose binding domains.
Additionally we removed the N-terminal targeting sequence from
Streptavidin as previous research had shown that the N-terminus
interrupts the binding of Streptavidin to Biotin. Flame retardants were
linked to a 6XHIS tag using a serine spacer. And then linked via the
same serine spacer to a Dockerin. The cellulose binding domain from
Clostridium Thermocellum was linked to a Cohesion from Clostridium
Thermocellum using a Clostridium specific linker. Our constructs were
then created through a combination of Gibson Assembly and ordering full
plasmids from twist. Constructs created through Gibson Assembly were
transformed into NEB 5-alpha before being identified via colony PCR and
verified through full plasmid sequencing.

### Test

For initial testing stages we started by transforming our test
constructs into BL21DE3. We then grew up the strains containing the
RFP-DocIII plasmid and induced them with 1mM IPTG. We started with the
RFP-DocIII plasmid because it served as an easy test subject, because we
assumed that inducing expression of the RFP would cause the culture to
turn red. However, while our constructs grew we noticed a growth deficit
in the induced culture, which suggested increased metabolic stress on
the cells which could possibly suggest production of proteins. We did
not see a red color.

![Figure 1: Comparative expression analysis](https://static.igem.wiki/teams/5823/images/engineering/engineering-fig-1.avif)

*Figure 1. Comparative expression of OD600 levels of
induced vs. noninduced plasmids for Cellulose Binding Domain
conjugated to a Cohesion
(CBM) and Red Fluorescent Protein (RFP) conjugated to a DocIII*

In order to fully probe this question we grew up our RFP conjugated to
Dockerin as well as a regular RFP before streaking onto two plates, one
containing IPTG and required Antibiotic and then one without antibiotic.
These plates were then incubated overnight at 37C. Before being imaged
in the morning. The image in the morning showed that the regular RFP
cultures turned red in the presence of the IPTG. But the ones containing
the RFP\_Doc did not turn red in presence of the IPTG.

![Figure 2: Bacterial growth plates](https://static.igem.wiki/teams/5823/images/engineering/engineering-fig-2.avif)

*Figure 2. Bacterial growth plates containing RFP\_DocIII and
unconjugated RFP. Plated both in presence or absence of IPTG*

Suggesting that the dockerin was preventing the RFP from being
successfully expressed in the cell, and after further research we found
this is in fact the case (11). Interestingly, months later, we noticed
that a pellet from an induced culture of the strain containing
RFP\_DocIII had turned red. Even though it was not red to begin with.
Given that insoluble proteins can slowly leak out of E.coli cells, this
could suggest that the reason for the lack of color from the RFP\_DocIII
could be that the protein is insoluble. When we attempted to purify the
pET28a we got very low yields.

### Learn

**Protein:** These two results suggested that our fusion
proteins attached to a dockerin would not express in either an easy or
efficient manner, so it was thus necessary to redesign our product. For
the next iteration we would thus design our proteins without the
dockerin. **DNA:** Although we observed differences in the
burning time between the treated and untreated samples, weighing the
samples before burning revealed that our drying time was far too short
and so it was necessary to prep our samples a full day ahead of when we
wanted to burn. We also decided to create a furnace with a live
temperature readout in order to get more accurate results. Additionally
in order to get more material we decided to transform a higher copy
number plasmid into the cells.

## DBTL #2

---

### Design

Our design stayed essentially the same, all we did was remove the
dockerins from the flame retardant candidates. The flame retardant
candidates were linked to a 6xHIS tag to aid in purification before
being linked to a cellulose binding domain via a serine spacer.
Additionally, for this iteration we elected to drop the optimized
Ribosome binding site in favor of a regular pET Ribosome Binding Site.
This is to reduce complexity and ensure uniform expression.

### Build

Our constructs were assembled using the optimized sequences. With the
same spacers as before. We then ordered optimized sequences from Twist
in a full plasmid format and transformed the full plasmid into a BL21DE3
expression strain.

### Test

For the testing of our new parts we first started with verifying
expressions. We did this by performing a protein growth curve on the
samples. However, as you can see from the gel below, it was difficult to
determine if there was any protein being expressed.

![Figure 3: Initial SDS PAGE with ambiguous results](https://static.igem.wiki/teams/5823/images/engineering/engineering-fig-3.avif)

*Figure 3. Initial SDS PAGE with ambiguous results*

After running multiple SDS-Page gels in this format we decided to run a
colony PCR on our transformed strains to ensure that the DNA was in fact
in our cells.

![Figure 4](https://static.igem.wiki/teams/5823/images/engineering/engineering-fig-4.avif)

*Figure 4. Gel from the colony PCR on our strains containing our
samples. From left: Ladder, Alpha-S-Casien, Alpha-S-Casein,
Streptavidin-1, Streptavidin-2, SBG-1, SBG-2, PurA-1, PurA-2,
pET28a-1, pET28a-2*

Finally we realized that it was necessary to normalize to a standard
OD600 value of 10 when we prepped our samples for the SDS-Page Gels.
This prevents the SDS from creating big streaks.

![Figure 5](https://static.igem.wiki/teams/5823/images/engineering/engineering-fig-5.avif)

*Figure 5. SDS-Page gel using samples from growing our samples
Alpha-S-Casein\_CBM and BetaLactoglobin\_CBM. From Left:
BetaLactoglobin\_CBM Not Induced, BetaLactoglobin\_CBM 1 Hr post
induction, BetaLactoglobin\_CBM 2 HR post induction,
BetaLactoglobin\_CBM 3 Hr Post induction, Alpha-S-Casein\_CBM 1 HR
post induction, Alpha-S-Casein\_CBM 2 HR post induction, 3 Hr Post
induction, Alpha-S-Casein\_CBM NI 3 HR post Induction (Different
day), Alpha-S-Casein\_CBM I 3 HR post Inducton (Differnt day),
BetaLactoglobin\_CBM NI 3HR post induction (Differnt day),
BetaLactoglobin\_CBM I 3HR post induction (Differnt day), Purified
Alpha-S-Casein\_CBM, Purified Betalactoglobin\_CBM,
BetaLactoglobin\_CBM only soluable, Alpha-S-Casein\_CBM only soluable.
From this we can visualize expression of the BetaLactoglobin, and
also confirm that the betalactoglobin is soluable.*

![Figure 5](https://static.igem.wiki/teams/5823/images/engineering/pura-gel.avif)

*Figure 6. SDS-Page gel using samples obtain from growing PurA\_CBM
and two other e.coli strains.
From Left: DH5a 1 HR, DH5a 2 HR, DH5a 3 HR, DH5a 4 HR,
DH5a 4HR, MG1655 1 HR, MG1655 2 HR, MG1655 3 HR, MG1655 3 HR,
MG1655 3 HR, MG1655 4 HR, PurA\_CBM 1 HR (Induced), PurA\_CBM 3 HR (Induced)
PurA\_CBM 3 HR (Induced and spun). From this we may be able to confirm expression of the
PurA\_CBM complex, there is a distinct band in the 3 HR sample that is not present in the 1 HR sample.
This also suggests that PurA\_CBM is soluable in E.coli which is encouraging for ease of expression.*

Once this was implemented we were able to confirm the expression of the
Beta-Lactoglobin CbpA construct. However, it appears that our
Alpha-S-Casein is not expressing. Additionally, as evidenced by the two
lanes that contain the purified protein, it appears that our
purification protocol needs modification.

**DNA:** We tested three different conditions, 0.625% w/v
DNA, 1.25% w/v DNA and 2.5% w/v DNA. Upon testing of our samples we
observed that the 2.5% w/v sample was able to resist burning at 400C for
three minutes.

### Learn

For future steps we will be sure to run all SDS-Page gels using the
similar format as above, including normalizing to a specific OD600 in
order to prevent SDS failure. Additionally we would like to perform
iterative design on our linkers in order to optimize for expression in
E.coli.

## Testing Flameretardancy Round 1

---

### Testing Flame Retardancy Round 1

This experiment investigated the effect of DNA treatment on the flammability of cotton samples
using a furnace-based burning test. Two conditions were tested: untreated cotton (0% w/v DNA)
and cotton treated with a 2.5% w/v DNA solution. Each sample was weighed before and after burning
to determine mass loss and observed during combustion to record temperature and burning behavior.
The untreated cotton ignited and burned rapidly, fully combusting within about 80 seconds, while
the DNA-treated cotton exhibited delayed ignition, slower burning, and greater structural integrity,
even after five minutes in the furnace. These results suggest that DNA treatment enhances flame resistance
and thermal stability, indicating its potential as a bio-based flame retardant. However, more testing
needs to be done in order to confirm these results and control the temperature measurements will help
quantify performance more precisely.

### Conditions Tested

| Condition Number | Water (uL) | DNA (uL) | % W/V DNA |
| --- | --- | --- | --- |
| 1 | 560uL | 0 | 0 |
| 2 | 452.2 | 107.8 | 2.5 |

Table 1: DNA coating conditions

Table 1 shows the DNA concentrations for our 2 conditions. Condition 1 was only water with 0% w/v DNA. Condition 2 was 2.5% w/v DNA. The solutions were dotted onto their respective pieces of cotton with a pipet.

## Mass of cotton before and after burning

Note: After taking initial measurements the cotton was cut in half in order to fit in the furnace

### Conditions Tested

| Condition Number | Mass before Burning (g) | Mass after Burning (g) | % of Cotton that Remains | % of Cotton that burned away | Notes |
| --- | --- | --- | --- | --- | --- |
| 1 | 0.2461 | 0.0176 | 7.15 | 92.85 | 1 piece was completely burnt away, so the mass after burning is just 1 burned piece |
| 2 | 0.2630 | 0.0236 | 8.97 | 91.03 | 1 piece was completely burned away, so the mass after burning is just 1 piece |

Table 2. Mass of Cotton for each condition before and afer burning

The mass of the cotton sample for each condition was recorded before and after burning them in the furnace. Initially, each cotton sample was found to be too large to fit on the furnace stand, so each was cut approximately in half, resulting in two samples per condition. The first sample for each condition was left in the furnace until it was completely combusted, while the second sample was removed once it had turned entirely black to measure the remaining mass after partial burning. The post-burning mass for Sample #2 was recorded for both conditions. For Condition 1, 92.85% of the cotton (Sample #1 and #2) mass was lost, while for Condition 2, 91.03% of the cotton (Sample #1 and #2) mass was lost. Table 2 shows the initial and final masses for each condition and percent of cotton that remained and burned away after the experiment.

In the spirit of DBTL, we relized the burning until complete incereration resulted in results that were difficult to compare between eachother. Additionally we realized that the burning was performed at different starting temperatures which again made the data difficult to compare between

For the next iteration, we plan to measure the mass of each sample before and after a controlled burn, and then limiting the furnace exposure to exactly one minute. This approach will allow us to compare mass loss across samples without complete combustion.

## Results from Burning videos

Since the cotton was cut in half, each condition was tested twice, with four total tests conducted on the cotton samples.
Sample #1 of Condition 1 and Sample #1 of Condition 2 both burned away completely. Table 3 shows the results of the experiment.

| Sample Activities | Condition 1, Sample #1 | Condition 2, Sample #1 |
| --- | --- | --- |
| inserted | 410C at 0 seconds | 420C at 0 seconds |
| starts browning | 410C at 2 seconds | 428C at 2 seconds |
| Black | 425C at 25 seconds | 400C at 50 seconds |
| Smoking | 417C at 20 seconds | 400C at 50 seconds |
| Embers | 435C at 40 seconds | 397C at 60 seconds |
| Burned Away | 429C at 80 seconds | 419C at 100 seconds |

Table 3. Comparison of Samples #1 of the untreated (Condition 1) and DNA-treated (Condition 2) vegetation samples under equivalent furnace conditions

Sample #2 of Condition 1 and Sample #2 of Condition 2 were both taken out after burning to determine their mass and calulate the mass which burned away

| Sample Activities | Condition 1, Sample #2 | Condition 2, Sample #2 |
| --- | --- | --- |
| inserted | 370C at 0 seconds | 410C at 0 seconds |
| starts browning | 370C at 2 seconds | 408C at 25 seconds |
| Black | 386C at 40 seconds | 423C at 50 seconds |
| Smoking | 386C at 40 seconds | 413C at 120 seconds |
| Embers | N/A | N/A |
| Removed | 400C at 120 seconds | 430C at 300 seconds |

Table 4. Comparison of the second sample of the untreated and DNA-treated samples.

To assess the performance and the effect of DNA as a flame retardnat,
we compared untreated plant samples (condition 1) to treated ones (Condition 2).
Howeverm because some samples were inserted into the furnance at different starting temperatures,
only Condition1 Sample #1 and Condition 2 sample #2 were compared to ensure consistency across datasets.
Both samples were introduced into the furnace at approximately 410°C, with temperature fluctuations remaining within 410°C–430°C throughout the trials. Table 5 below shows a comparison of the aforementioned conditions.

| Sample Activities | Condition 1, Sample #1 | Condition 2, Sample #2 |
| --- | --- | --- |
| inserted | 410C at 0 seconds | 410C at 0 seconds |
| starts browning | 410C at 2 seconds | 408C at 25 seconds |
| Black | 425C at 25 seconds | 423C at 50 seconds |
| Smoking | 417C at 20 seconds | 413C at 120 seconds |
| Embers | 435 at 40 seconds | N/A |
| Removed | 429C at 80 seconds | 430C at 300 seconds |

Table 5. Comparison of untreated (Condition 1) and protein-treated (Condition 2) vegetation samples under equivalent furnace conditions.

The untreated sample (Condition 1) exhibited rapid combustion behavior: visible browning occurred within 2 seconds of insertion, followed by blackening and smoke production within 25 seconds. Embers appeared shortly after, and the sample was fully consumed in approximately 80 seconds.

In contrast, the protein-treated sample (Condition 2) showed significantly delayed combustion. Browning did not begin until 25 seconds, and visible smoke was not observed until around 120 seconds. Notably, the treated sample did not produce embers, retained structural integrity throughout, and required removal after five minutes at similar temperatures due to minimal burning.

These results demonstrate that the DNA substantially increased the sample’s resistance to ignition and thermal degradation. The delayed onset of browning and smoke, along with the absence of embers, indicates improved thermal stability and reduced flammability. Moving forward, repeating these trials with additional replicates and temperature-controlled measurements will help quantify performance more precisely. Nonetheless, this experiment highlights the furnace’s effectiveness as a reliable testing platform and validates the protein’s potential to enhance vegetation fire resistance.

Finally for next time, in addition to burning for a set time and ensuring consistent start temperature we will also work to fully submerge our cotton in the DNA solution. Our current protocol involves dotting the solution on with a pipette tip which could lead to inconsistent spreading of the sample and control, and this may explain the spotty results

## References

---

1. Alongi, J.; Poskovic, M.; P.m., V.; Frache, A.; Malucelli, G.
   Cyclodextrin Nanosponges as Novel Green Flame Retardants for PP, LLDPE
   and PA6. Carbohydrate Polymers 2012, 88 (4), 1387–1394.
   <https://doi.org/10.1016/j.carbpol.2012.02.038>
2. Bourbigot, S.; Le Bras, M.; Duquesne, S.; Rochery, M. Recent Advances
   for Intumescent Polymers. Macromolecular Materials and Engineering
   2004, 289 (6), 499–511.
   <https://doi.org/10.1002/mame.200400007>
3. Ntuli, T.; Xaba, M. S.; Ntsendwana, B.; Mokhena, T. C.; Mochane, M. J.
   Recent Advances in Protein-Based Flame-Retardant Materials. In
   Biomaterials as Green Flame Retardants; Mokhena, T. C., Mochane, M.
   J., Sadiku, E. R., Sinha Ray, S., Eds.; Springer Nature: Singapore,
   2024; pp 285–316.
   <https://doi.org/10.1007/978-981-97-6871-4_12>
4. Alongi, J.; Cuttica, F.; Blasio, A. D.; Carosio, F.; Malucelli, G.
   Intumescent Features of Nucleic Acids and Proteins. Thermochimica Acta
   2014, 591, 31–39.
   <https://doi.org/10.1016/j.tca.2014.06.020>
5. Etzel, M. R. Manufacture and Use of Dairy Protein Fractions. The
   Journal of Nutrition 2004, 134 (4), 996S-1002S.
   <https://doi.org/10.1093/jn/134.4.996S>
6. Wang, Y.; Liu, C.; Lai, J.; Lu, C.; Wu, X.; Cai, Y.; Gu, L.; Yang, L.;
   Zhang, G.; Shi, G. Soy Protein and Halloysite Nanotubes-Assisted
   Preparation of Environmentally Friendly Intumescent Flame Retardant
   for Poly(Butylene Succinate). Polymer Testing 2020, 81, 106174.
   <https://doi.org/10.1016/j.polymertesting.2019.106174>
7. Alongi, J.; Carletto, R. A.; Blasio, A. D.; Carosio, F.; Bosco, F.;
   Malucelli, G. DNA: A Novel, Green, Natural Flame Retardant and
   Suppressant for Cotton. J. Mater. Chem. A 2013, 1 (15), 4779–4785.
   <https://doi.org/10.1039/C3TA00107E>
8. Daniel, D. C.; Johnson, E. M. PURA, the Gene Encoding Pur-Alpha,
   Member of an Ancient Nucleic Acid-Binding Protein Family with
   Mammalian Neurological Functions. Gene 2018, 643, 133–143.
   <https://doi.org/10.1016/j.gene.2017.12.004>
9. Bayer, E. A.; Belaich, J.-P.; Shoham, Y.; Lamed, R. The Cellulosomes:
   Multienzyme Machines for Degradation of Plant Cell Wall
   Polysaccharides. Annual Review of Microbiology 2004, 58 (Volume 58,
   2004), 521–554.
   <https://doi.org/10.1146/annurev.micro.57.030502.091022>
10. Goldstein, M. A.; Takagi, M.; Hashida, S.; Shoseyov, O.; Doi, R. H.;
    Segel, I. H. Characterization of the Cellulose-Binding Domain of the
    Clostridium Cellulovorans Cellulose-Binding Protein A. J Bacteriol
    1993, 175 (18), 5762–5768.
    <https://doi.org/10.1128/jb.175.18.5762-5768.1993>
11. Brás, J. L. A.; Carvalho, A. L.; Viegas, A.; Najmudin, S.; Alves, V.
    D.; Prates, J. A. M.; Ferreira, L. M. A.; Romão, M. J.; Gilbert, H.
    J.; Fontes, C. M. G. A. Escherichia Coli Expression, Purification,
    Crystallization, and Structure Determination of Bacterial
    Cohesin-Dockerin Complexes. Methods Enzymol 2012, 510, 395–415.
    <https://doi.org/10.1016/B978-0-12-415931-0.00021-5>

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your project. It is designed to provide sufficient information for other teams to replicate our work.{% endblock %}
{% block page\_content %}

# Experiments

##### Navigation

[Molecular Biology Techniques](#molecular-biology)

[1.Gibson Assembly Protocol](#gibson-assembly-protocol)
[2.Polymerase Chain Reaction (PCR)
Protocol](#pcr)
[3.Colony Polymerase Chain Reaction
(cPCR) Protocol](#colony-pcr)

[Cloning and Transformation](#cloning-transformation)

[1.Making Overnight Cultures Protocol](#overnight-cultures)
[2.Plasmid Preparation Protocol](#plasmid-prep)
[3.Transformation Protocol](#transformation)

[Gel Based Techniques](#gel-techniques)

[1.DNA Gel Electrophoresis Protocol](#dna-gel)
[2.Sodium Dodecyl
Sulfate-PolyAcrylamide Gel Electrophoresis (SDS-PAGE) Gel
Protocol](#sds-page)
[3.Making Agar Plates Protocol](#agar-plates)

[Analytical Techniques](#analytical)

[1.Protein Purification](#protein-purification)
[2.Protein Growth Curve Protocol](#protein-growth)
[3.Burning Assay Protocol](#burning-assay)

## Molecular Biology Techniques

---

#### Gibson Assembly Protocol

1. Design fragments (G-Blocks) and order the necessary primers to open
   destination plasmid and prep plasmid for Gibson Assembly.
2. Digest with DPN1 to remove the backbone (1uL for overnight).
3. Perform Gibson Assembly Reaction.
   **Note:** All
   components should be kept on ice.

| Components | Amount |
| --- | --- |
| Fragments (G-Blocks) | 2 µL of each fragment |
| Plasmid Vector | 0.02 - 0.5 pmoles of vector |
| 2x Gibson Assembly Master Mix | 10 µL |
| Mili-Q Water | Adjust to final volume of 20 µL |
| Total Volume | 20 µL |

4. Incubate in a thermocycler at 50ºC for 1 hour.
   **Note:**
   Time of incubation will vary based on size and number of fragments.
5. Run on agarose gel to check size.
6. Transform into competent E.Coli cells.

#### Polymerase Chain Reaction (PCR) Protocol

##### Primer Reconstitution

1. Spin lyophilized primer vials at 6800G for 3 minutes.
2. Resuspend primers to a final concentration of 100nM.
3. Prepare 10 nM stock of both forward and reverse primers by diluting
   with DI water.

##### Prepare 50 µL PCR Reaction Mix

Follow the table for making the PCR reaction mix.

| Component | Amount | Final Concentration |
| --- | --- | --- |
| Q5 2X Master Mix | 25 µL | 1X |
| Forward Primer | 2.5 µL | 0.5 µM |
| Reverse Primer | 2.5 µL | 0.5 µM |
| Template DNA | Variable | ~50-100ng |
| DI Water | To 50 µL | -- |

##### Amplification of Desired Segment using Thermocycler

Follow the table to amplify the DNA segment with a Thermocycler.

|  | Temperature | Time (s) |
| --- | --- | --- |
| **Denaturation (1):** | 94°C | 30 |
| **Denaturation (2):** | 94°C | 5 |
| **Anneal\*:** | 60-72°C | 25 |
| **Extend (1):** | 72°C | 30 seconds/ Kb |
| **Cycle Number:** | 28 |  |
| **Extend (2):** | 72°C | 5 minutes |
| **Hold:** | 4°C | Indefinitely |

**\*Will vary depending on primer**

##### Purification and Quantification

1. Purify using Monarch PCR Purification Kit.
2. Elute 30 µL into an eppendorf tube.
3. Nanodrop purified product by adding 1 µL of the sample.
4. Record concentration (ng/µL), A260/A280 score, and A260/A230 score.

   **Note:** Blank nanodrop first with the elution
   buffer to calibrate.

#### Colony Polymerase Chain Reaction (cPCR) Protocol

##### Primer Reconstitution

1. Spin lyophilized primer vials at 6800G for 3 minutes.
2. Resuspend primers to a final concentration of 100nM.
3. Prepare 10 nM stock of both forward and reverse primers by diluting
   with DI water.

##### Pick Colonies

1. Add 100 µL of nuclease free water to an eppendorf tube.
2. Pick large, well isolated single colonies and inoculate nuclease free
   water.

##### Prepare 50 µL cPCR Reaction Mix

Follow the table for making the cPCR reaction mix.

| Components | Amount |
| --- | --- |
| One Taq 2x Reaction Buffer | 25 µL |
| Forward Primer | 1 µL |
| Reverse Primer | 1 µL |
| Template DNA | 10 µL |
| DI Water | 13 µL |
| Total | 50 µL |

##### Amplification of Desired Segment using Thermocycler

Follow the table to amplify the DNA segment with a Thermocycler.

|  | Temperature | Time (s) |
| --- | --- | --- |
| **Denaturation (1):** | 94°C | 30 |
| **Denaturation (2):** | 94°C | 5 |
| **Anneal:** | 46°C\* | 25 |
| **Extend (1):** | 68°C | One minute / Kb |
| **Cycle Number:** | 30 |  |
| **Extend (2):** | 68°C | 300 |
| **Hold:** | 4°C | Indefinitely |

**\*Will vary by primer**

##### Run Product on DNA Gel

1. To determine if appropriate DNA was inserted and amplified, run cPCR
   reaction on DNA Gel following DNA Gel Electrophoresis Protocol.

## Cloning and Transformation

---

#### Making Overnight Cultures Protocol

##### Plate

1. Pick one colony from the chosen transformation plate using a 10 µL
   micropipette tip.
2. Place the micropipette tip into an eppendorf tube containing 100 µL of
   nuclease free water to resuspend the colony.
3. Add 5 mL of LB broth to a 15 mL Conical tube along with the
   appropriate antibiotics.
4. Take 50 µL of the nuclease-free water and colony mixture and add it
   into the 15 mL conical tube.
5. Take the other 50 µL of the water and colony mixture and plate it on
   the appropriate antibiotic plate.
6. Store the overnights and plates on the 37ºC.
7. Place all leftover water and colony mixture into the fridge. Labeled
   with the appropriate colony designation and the date.

##### Glycerol Stock

1. Add 5 mL of LB Broth plus antibiotics to a 15 mL conical tube.
2. Using a 5 mL glass pipet, stab the glycerol stock and transfer a shard
   of the stock to the LB.
3. Incubate overnight at 37ºC.

#### Plasmid Preparation Protocol

**Following Monarch Spin Plasmid Miniprep Kit from New England
BioLabs**

1. Pellet 1-5 mL (not to exceed 15 OD units) bacterial culture by
   centrifuging at 16000G for 30 seconds. Discard supernatant.
2. Resuspend pellet in 200 μL of B1 Resuspension Buffer. Vortex to ensure
   suspension.
3. Add 200 μL of B2 Plasmid Lysis Buffer and invert. Let sit at room
   temperature for one minute.
4. Add 400 μL of B3 Neutralization Buffer, invert a few times, and let
   sit at room temperature.
5. Centrifuge at 16000G for 3 minutes.
6. Transfer supernatant to labeled spin column and centrifuge again at
   16000G for 1 minute. Discard flow-through.
7. Re-insert the column into the collection tube and add 200 μL of
   Plasmid Wash Buffer 1. Centrifuge at 16000G for one minute and discard
   flow-through.
8. Add 400 μL of Plasmid Wash Buffer 2 and centrifuge for one minute.
9. Transfer column to a clean 1.5 mL micro-centrifuge tube.
10. Add 30 μL of DNA Elution Buffer to the center of the matrix, and then
    wait one minute before centrifuging at 16000G for 1 minute.
11. Use the NanoDrop machine to check the concentration of DNA in the
    final eluted DNA.

#### Transformation Protocol

1. Thaw 50 µL of competent BL21DE3 cells and add 2 µL of DNA into
   competent cells. Incubate on ice for 30 minutes.
2. Heat shock mixture in heat-block at 42ºC for 30 seconds and put back
   on ice for 2 minutes.
3. Preheat Super Optimal Media with Catabolite Repression (SOC) media to
   37ºC. Add 950 µL of SOC to a final volume of ~1000 µL.
   **Note:**
   This step should be done in a sterile Biosafety Cabinet (BSC)
4. Incubate at 37ºC for 60 minutes. During this incubation period there
   needs to be aeration of some kind. For this, cells were put on a shake
   plate at 300 RPM.
5. Dry three LB plates with appropriate antibiotics in an incubator set
   to 37ºC. Plate 100 µL onto one plate and then plate 200 µL onto each
   of the remaining plates. Spread evenly around plate using a Colony
   Spreader.
6. Incubate at 37ºC overnight.

## Gel Based Techniques

---

#### DNA Gel Electrophoresis Protocol

##### Make Gel

1. To make a gel add 1G of agarose to 100 mLs of 1X TAE in a glass
   beaker.
2. Microwave on high until dissolved. The solution should appear clear,
   if there is any streakiness or cloudiness keep microwaving.
3. For 100 mLs usually takes about 3 minutes.
4. Remove from the microwave with thick orange gloves and set to cool
   until you can comfortably touch with a bare hand.
5. Once you can comfortably touch the beaker with your bare hands, add in
   1 μL of SYBR Safe stain for every 10 mLs of solution you have.
6. A 50 mL gel would get 5 μL of SYBR Safe.
7. Add a comb to a gel box and pour the gel into the gel box.
8. Make sure the gel box is assembled in such a way that the gel will not
   run out if you pour it in. The gel apparatus consists of a big open
   box with two electrodes and a smaller box with rubber seals and open
   on both ends. Make sure that the smaller box is put in on the elevated
   platform of the bigger box and with the open ends facing the walls of
   the bigger box. You should have to push hard as the rubber seals will
   drag.
9. When pouring the gel make sure that the gel covers the entire bottom
   of the box, if there are any open spaces, you have to add more.
10. Finally, when pouring the gel try to limit the number of bubbles in
    the path of the DNA.
11. If there are bubbles use a pipette tip to move them to the side.
12. Once gel has solidified (should be firm to touch), remove gel from
    orientation and rotate it 90 degrees. Remove the comb and pour in 1X
    TAE.
13. Make sure the gel is oriented correctly (as in the long part of the
    gel is facing towards the positive electrode).
14. Cover with enough 1X TAE so that the gel is covered, ensuring that the
    buffer goes into the wells.

##### Prep Samples

1. Portion out sample into a separate PCR tube.
2. Usually 5 uL of sample.
3. Add loading dye and mix thoroughly by pipetting up and down.
4. Loading dye will typically be at 6X concentration, so add 1 uL of dye
   to 5 uL of sample.
5. Create gel legend to keep track of the locations of the ladder and
   samples.
6. Make descriptive names so someone coming after you can understand
   which lane each sample corresponds to.

##### Load Gel

1. Load ladder and samples from left to right to ensure that you don't
   miss a sample or get confused.
2. Load 3 uL of ladder.
3. Load 6 uL of the samples.
4. Go in with the pipette very slowly, careful not to puncture the bottom
   of the gel.
5. Do not go past the second stop, this will expel air into the well and
   ruin your samples.
6. It can be helpful to use the other hand to steady the hand with the
   pipette
7. If your samples float away then you either need to add more loading
   dye or did not mix it sufficiently
8. To confirm ladder and samples were loaded properly, there should be a
   semi solid line of color at the bottom of the well.

##### Run Gel

1. Cover tank with gel cap.
2. There will be two wires coming out of it.
3. Make sure that the negative electrode is closest to the lanes.
4. Plug the tank into the power bank and run at 150 V for 30 minutes.
5. You can check that everything is working by seeing if bubbles appear
   in the 1X TAE.

##### Visualize Gel

1. Remove gel from box and place gel on gel imager to image.
2. Make sure that the gel imager is using the proper stain.
3. Export image and save.

##### Clean Up

1. The 1X TAE can be reused, so pour it into a 1 L bottle and label with
   the number of times it has been used.
2. Throw the gel away into biohazard waste.
3. If you accidentally made too many gels:
   Place the extra gels
   into a plastic bag and label with name, date, and gel stain +
   concentration (SYBR Safe, 1% Agarose).
   Add a little bit of 1X
   TAE into the bag.
   Store at 4°C.

#### Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis (SDS-PAGE) Gel Protocol

##### Sample Preparation

1. Combine the samples with 50 µL 10% PBS and 50 µL Laemmli.
2. Make Laemmli + PBS + B-mercapnoethanol mix by:
   50/50 of 10% PBS
   and 2x Laemmli + 5% B-mercapnoethanol
3. Resuspend samples in 100 uL of Laemmli mixture.
4. Place samples on a heat block for 10 minutes at 95°C.
5. Vortex all samples for 5 minutes.

##### Gel Set Up

1. Obtain Invitrogen Gel:
2. Remove packaging of gel.
3. Remove tape from the bottom of the gel.
4. Remove the comb from the top of the gel.
5. Rinse gel thoroughly with DI water.
6. Carefully insert gel into the tank.
7. Fill up the tank with 1x SDS buffer until the fill line.
8. Load ladder and samples carefully.
9. Run gel for 1 hour at 150V.

##### Staining

1. Remove gel from the tank carefully and remove from packaging
2. rinse several times in DI water.
3. Add 50 mL of Coomassie stain to the gel and cover with tin foil and
   leave it to stain overnight while rocking.

#### Making Agar Plates Protocol

1. Prepare LB Agar media. Mix LB Agar (Miller) power with DI water.
   Sigma-Aldrich LB Broth with Agar powder was used for plates.

   **Note:** 40g of LB agar powder per 1 liter of DI
   water.
2. Sterilize and dissolve LB-Agar mixture by microwaving in 30 second
   increments until powder is completely dissolved, swirling container
   between each increment until clear.
   **Note:**
   Autoclaving can also be used for this step: 20 min at 15 psi (1.05
   kg/cm2) on liquid cycle.

**The following steps should be done in a sterile Biosafety Cabinet
(BSC):**

3. Allow mixture to cool for 5 minutes and add sterile antibiotic, swirl
   container to mix:

| Antibiotic | Recommended Stock Concentration (mg/mL) | Recommended Working Concentration (µg/mL) |
| --- | --- | --- |
| Ampicillin | 100 | 100 |
| Kanamycin | 50 | 50 |

5. Before LB Agar mixture is completely cool, pipette 12 mL of mixture
   into each plate. Allow plates to set without lids on to avoid
   condensation.
6. Plates can be stored at 4ºC until use.

## Analytical Techniques

---

#### Protein Purification Protocol

##### Prep the Cellulose

1. Add Microcrystalline Cellulose (0.2g), along with distilled water
   (0.6mL). Mix lightly in beaker in order to create a slurry.

   **Note:** The cellulose will not dissolve, just
   create a slurry.
2. Slowly add 86% Sulfuric Acid (10 mL) with stirring. Let the solution
   sit on ice for 1 hour with occasional stirring.
3. Add 40 mL of ice-cold water gradually with stirring. This should
   result in a whitish, cloudy precipitate.
4. Using a swing bucket rotor, centrifuge the cellulose at 3,000G for 10
   min at 4°C.
5. Decant the supernatant.
6. Resuspend in cold water and then centrifuge again at 3,000G for 10 min
   at 4°C.
7. Repeat the previous step four times.
8. If needed you can resuspend in 45 mLs of DI water and store at 4°C.
9. After the final wash measure the pH of the solution and if it is under
   5 resuspend in 0.5 mLs of Na2CO3 (2M), along with 45 mLs of water.
10. Centrifuge at 3,000G for 10 minutes at 4°C and resuspend in DI water.
11. Remeasure the PH of the solution and repeat 9 and 10 until the
    solution reaches a pH of 5-7.
12. Resuspend the final solution in 40 mLs of DI water and store at 4°C.

##### Protein Purification

1. Transfer cultures into 50 mL conical tubes.
2. Harvest culture by spinning at 6800G for 5 minutes.
3. Decant supernatant.
4. Resuspend each culture in 15 mLs of 0.5% Tween20.
5. Place the conical tube in an incubator to shake for 10 minutes.
6. Combine 10 mLs of the NaOH + cellulose solution with 10 mLs of the
   lysate solution in a beaker with a stir bar.
7. Place the beaker on a stir plate for 10 minutes.
8. Transfer to another 50 mL conical tube and spin down at 14000G or max
   speed for 10 minutes.
9. Add 10 mLs of ethylene glycol and shake in hand for 2 minutes.
10. Spin down again at 14000G for 10 minutes.
11. Transfer supernatant into fresh 50 mL flasks and store at -20°C with
    the correct label.

#### Protein Growth Curve Protocol

1. Take cultures out of the incubator at 37°C.
2. Make flasks with 47.5 mLs of LB and 50 µL of Kan50.
3. Add 2.5 mLs of cultures to each of the flasks.
4. Put all the flasks in the 37°C incubator.
5. Grow until an Optical Density at 600 nanometers (OD600) of 0.6.
6. Induce half of the flasks with 1 mM IPTG and the other half with 250
   µM IPTG.
7. Place half of the flasks in at 32°C.
8. Grow for three hours and record OD600.
9. Take 1 mL samples every hour to make protein growth curves.
   * X-axis: Time (Minutes)
   * Y-axis: Optical Density at 600 nanometers (OD600)

#### Burning Assay Protocol

##### Prep the DNA

1. Plasmid prep the DNA and elute in water.
2. Nanodrop the DNA.
   * Record concentration (ng/µL), A260/A280 score, and A260/A230
     score.
3. If multiple preps were completed, combine all into one vial and
   calculate the new concentration and the total amount of DNA,
4. In fresh eppendorf tubes, create four different conditions:
   * Water
   * 0.625% by weight DNA
   * 1.25% by weight DNA
   * 2.5% by weight DNA
5. For example; if the DNA solution was 92.4 ng/μL
   * Condition 1: 560 μL water
   * Condition 2: 26 μL DNA solution -> 534 μL of Nuclease free water
   * Condition 3: 53 μL DNA solution -> 507 μL of Nuclease free water
   * Condition 4: 107.8 μL DNA solution -> 452.2 μL of Nuclease free
     water

##### Apply to Cotton

1. Take cotton and cut into strips ⅝ inch wide.
2. Weigh the cotton before adding.
3. Holding cotton with a pair of plies with one hand use a pipette to dot
   the DNA solution onto the cotton swab, adding just a thin layer. Cover
   both sides with the solution.
4. Place in a petri dish and label and leave on the bench overnight to
   dry.

{% endblock %}