iGEM 2023 — Team asij-tokyo

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Unable to display PDF file. [Download](https://static.igem.wiki/teams/4738/wiki/asij-tokyo-notebook.pdf) instead.

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{% block title %}Engineering Success{% endblock %}
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Throughout the duration of the project, our team continuously referred to the established principles of the
engineering cycle: design, build, test, and learn. Our approach to developing an efficient PHB secretion
system in E. Coli consisted of two main stages, each of which builds up the specific functions of our design
and acts as a progressive checkpoints for our project.

#### Phasin Secretion System

Verification and evaluation for the secretion of the phasin protein in recombinant E. Coli. using type I
secretion system (HlyA), type II secretion system (GeneIII & TorA), and vescile nucleating peptides (VNps).

#### PHB Secretion System

An improved design of the phasin secretion system: Verification and evaluation for the secretion of the PHB
in recombinant E. Coli. using type I secretion system (HlyA), type II secretion system (GeneIII & TorA), and
vescile nucleating peptides (VNps).

In our attempts to develop our intended model for an improved PHB secretion system, we encountered challenges
on several occasions. By adhering to the design cycle, practicing accurate measurements, and carefully
documenting our work, we were able to gain a better understanding of the functionalities behind PHB
production as well as a successful secretion system. We hope our work and cataloged information will provide
valuable insight for any future research into PHAs and secretion in bacteria.

# STAGE ONE: Phasin Secretion System

## Iteration 1

### Design

Through preliminary research and literature review, we identified phasin as a target protein for the indirect
secretion of PHB. We designed initial model for phasin production to consist of a protein encoding gene,
PhaP. For our experimental secretion model, we combined the phasin production model with an HlyA tag for
type I secretion. We incorporated a His6 tag to provide us the ability to run protein purification in later
steps of quantification and measurement. Our constructs are listed below:

![](https://static.igem.wiki/teams/4738/wiki/en1.png)

This construct is comprised of T7 Promoter/lacO, RBS, Phasin, His6, T7 Terminator, and the plasmid backbone
(pSB1A3). It is designed for the E. coli to be able to produce phasin.

![](https://static.igem.wiki/teams/4738/wiki/hlya-his-v1.png)

This construct is comprised of T7 Promoter/lacO, RBS, HlyA, Phasin, His6, T7 Terminator, and the plasmid
backbone (pSB1A3). It is designed for the E. coli to be able to both produce phasin as well as
simultaneously undergo secretion.

We considered how we may expand on the work of Calgary 2022’s iGEM team and test various RBSs. In addition to
that, we knew we would be testing various secretion tags. Therefore, we ordered each part as its own
fragment for Gibson Assembly.

### Build

We assembled our two groups via Gibson Assembly, followed by a standard transformation protocol with BL21
(DE3) E. Coli. We incorporated an additional negative control group consisting of untransformed E. Coli. Our
team utilized ampicilin and chloramphenicol to confirm successful transformation of our cultures.

Then the groups were separated into two samples. The first groups were induced by IPTG to initiate the
production of phasin and allowed to inclubate for an additional 3 hours while the second were remained
uninduced.

### Test

In order to pellet cells to separate intracellular and extracellular components of the cell culture, samples
of the IPTG-induced groups were centrifuged at high speeds. Once isolated, the pelleted E. coli cells were
lysed to obtain intracellular components which includes proteins of interest.

After cell cysis, samples of each of the intracellular and extracellular components were spun in nickel
column tubes. We made sure to collect and preserve samples in each of the wash, bind, and elute steps.

### Learn

We realized that we had misunderstood how to design parts for Gibson assembly on the days following protein
purification. We had assumed that Gibson Assembly would be compatible with gene fragments with very low
number of base pairs such as our RBS but we learned that it is only optimal for fragments with length 200+,
with shorter fragments requiring additional steps which we had not taken. We could not find any literature
helpful to our specific requirements, to be able to interchange any part of the construct where certain
parts by be as small as 15 bp, and since designed primers for Gibson Assembly surpassed such parts in size,
we concluded that Gibson Assembly would not work for our situation.

Additionally, after conducting our first lab that we planned ourselves, we realized we were greatly
underprepared and this resulted in unorganized, underdocumented outcomes.

We identified the following factors as points of failure:

* Lab members swapping midway through the procedure
* Lack of documentation and labeling expectations
* Underestimating resources and time required for the lab

## Iteration 2

### Design

After our first iteration for designing a phasin production and secretion system, we revised our parts to be
compatible with Golden Gate Assembly. We reasoned that Golden Gate Assembly would be compatible with the
smaller parts and modularity requirements of our plan.

Furthermore, we incorporated an RFP flipper design into our original pSB1A3 plasmid backbone. The use of an
RFP flipper would allow us to indicate successful versus unsuccessful colonies in assembly.

In addition, we removed the IPTG induction component of our design. We found it to be redundant in the
context of maximizing the production of our target phasin protein. To do this, we simply replaced the
T7/LacO promoter with a T7 promoter part without the lac operon. Our revised constructs are visualized
below:

![](https://static.igem.wiki/teams/4738/wiki/ns-his-v2.png)
![](https://static.igem.wiki/teams/4738/wiki/hlya-his-v2.png)

### Build

Before assembling the parts, we inserted the RFP flipper into our pSB1A3 plasmid backbone to make the pSB1A3
backbone compatible with our assembly through BioBrick Assembly. This alsos allow for the integrated RFP
flipper design.

In our altered protocol, we utilized the NEBridge Golden Gate Assembly Kit for Golden Gate Assembly. We
followed the same previous procedures for transformation. This time, the cultures were not induced by IPTG.

### Learn

This iteration of the design cycle ended with complications in ordering parts which guaranteed our construct
was not correctly assembled. After transformation, we tried to confirm our parts were ordered to arrive with
the correct overhangs, specifically for the promoter and terminator in order to be compatible with the RFP
flipper and the RBS so we have 2 variants (one to ligate directly to the phasin coding fragment and another
to the HlyA coding fragment). Unfortunately, we were unable to obtain information on the exact sequence that
was synthesized based on our order and the lack of a second RBS variants when we received our order
confirmed we had at least one issue with the parts we got. At this point, we decided there wan’t a point in
continuing our lab as we cannot determine what exactly we constructed.

We considered designing parts with Type IIS restriction sites to generate the overhangs ourselves and
eliminate the possibility of errors with ordering, however with time considerations we had changed our plan
to not expand on RBS testing by iGEM team Calgary 2022 which made use rethink the benefits of modularity.

## Iteration 3

### Design

Due to consistent errors in our modes of assembly, we made the final adjustment to use BioBrick Assembly as
our main method for part assembly. Instead of managing a large number of individual parts and respective
primers, we also chose to simplify our sequence into single-composite parts. On top of that, we could reuse
these parts when our experiments progress to secreting PHB by inserting a PHB synthesizing construct after
our design for phasin secretion.

Additionally, in order to maximize efficiency with our experimentations, we selected additional models for
our secretion system to test all together. These additions include the type II secretion system via TorA and
GeneIII secretion tags. More information and background on these systems can be found in the extended
literature review. The constructs for each of these models are the following:

![](https://static.igem.wiki/teams/4738/wiki/tora-his-v1.png)
![](https://static.igem.wiki/teams/4738/wiki/geneiii-his-v1.png)

### Build

Through a restriction digest and a ligation step, BioBrick Assembly was carried out with the previously
listed parts and the pSB1A3 backbone. The assembled plasmid DNA were inserted into E. Coli cells via the
same basic transformation protocol.

### Test

Again, we performed cell lysis with the pelleted cells samples contraining intracellular components. Each one
of our samples were put through a nickel spin-column based purification step. And this time, we conducted
our first quantitative measurements of our protein concentration using a Bradford Assay. However, our
results with the initial Bradford Assay was inconclusive.

We followed up our Bradford Assay with an SDS-page analysis in order to confirm the success of our
purification step. These results, too, were found to be inconclusive.

### Learn

Based on our initial Bradford Assay results, absorbance readings indicated practically insignificant protein
quantities in all samples with slight differences between experimental groups. We came up with the following
as possible sources of error:

* Samples were overly diluted
* Our target proteins were lost in purification
* Wrong buffer was used to make the standard curve

To determine the exact cause of error, we first attempted to run an SDS-page analysis on samples throughout
the purification process to observe whether our target protein was lost in stages before the elute. However,
many lanes on our page didn’t travel even with prolonged time and the ones that did remained towards the
top.

Then, reran protein purification then Bradford Assay on a sample from the same batch with a couple of
modifications, specifically the amount of elution buffer was halved to increase protein concentration and
elution buffer from protein purification was used to produce the standard curve instead of PBS buffer, this
way conditions between the sample and standard are more similar. However, readings once again indicated
fairly low concentration of proteins across all groups.

We made another attempt at SDS-page with extra time spent denaturing the proteins as refolding may have been
a possible reason proteins failed to travel in the previous attempt. This time, we were able to get some
movement after leaving the proteins on the page for multiple additional hours. We were able to compare the
proteins in bind, wash, and elute stages of purification between the inside and outside portions of the
non-secreting group. Based on the existence of tall bands covering wide ranges of protein weights in all
elute solutions, it was indicated that our purification method wasn’t efficient in isolating our his-tagged
phasin. Furthermore, analysis of the outside group indicates a thick band assumed to be phasin only flowed
through the column for the elute stage but the same band in the inside group seems to flow through in both
the bind and wash stages. From this, we concluded that nickel spin columns with his-tagged proteins may not
be the optimal method to purify our target protein.

## Iteration 4

### Design

Based on what we learned from our previous efforts, we implemented appropriate changes to our protocol and
design. First, we used samples from a fresh new batch of E. Coli that had been continually incubating since
the day of transformation. These tubes had shown more cell growth than the initial round of testing.
Therefore, we assumed that these cells were more likely to have produced more proteins than in the previous
batch. Furthermore, we reduced dilution of the samples in order to provide us with higher concentration
values of proteins to work with.

Instead of testing all the groups, we chose three to work with in order to save resources and time while
minimizing error. These three groups were the non secretion group, type II secretion group via HlyA, and a
negative control consisting of E. Coli that had been growing in SOC solution.

A major change to our approach was the implementation of an alternative purification method. As previously
mentioned, one plausible error with our experimentation was the nickel spin column-based protein
purification step. We were not able to confirm its accuracy due to inconclusive SDS-page results. Therefore,
we utilized a different protein purification this time using hydrophobic interaction chromatography (HIC).

### Test

In testing phasin secretion systems, we conducted the improved versions of our experimental design including
HIC chromatography and Bradford Assay protein quantification.

### Learn

Although initial readings indicated a significant protein concentration from a high absorbance reading, we
realized this may partially be due to the presence of the dye used to show the flow of solution through the
HIC column. Unable to distinguish the actual readings for the proteins themselves, HIC chromatography was
not compatible with Bradford Assay protein quantification. Additionally, HIC chromatography with the kits we
had access to inherently doesn’t allow us to get consistent flow through, so comparing values between
experimental groups wasn’t going to be possible. For these limitations, we decided to move away from a
purify then quantify approach entirely.

## Iteration 5

### Design

At this point, we had encountered consistent errors and indecisive results twice, even after several attempts
of troubleshooting. To resolve these issues, we came up with a larger redesign of our experimental approach.
Instead of using a Bradford Assay and having to isolate our target phasin proteins, we decided on
implementing a GFP-reporter tag to each of our constructs.

By adding a GFP reporter tag to our phasin protein, we would not only be able to qualitatively assess the
production of our protein but be able to quantify its concentration values through fluorescent readings.

Nevertheless, it is important to identify the specific assumptions we made in implementing such design. These
various assumptions would need to be verified in future experiments.

1. GFP proteins will be produced as part of or in equal ratio to the phasin proteins.
2. GFP-tagged phasin proteins are produced similarly to that of non-GFP-tagged phasin proteins. In other
   words, the addition of a GFP tag has a minimal affect on the production rates and quality of phasin
   proteins.
3. GFP-tagged phasin proteins are secreted similarly to that of non-GFP-tagged phasin proteins. In other
   words, the addition of a GFP tag has a minimal affect on the efficiency of phasin protein secretion.

With such assumptions in mind, we redesigned our previous constructs to include the GFP tag as listed
below.

![](https://static.igem.wiki/teams/4738/wiki/ns-gfp.png)
![](https://static.igem.wiki/teams/4738/wiki/hlya-gfp.png)
![](https://static.igem.wiki/teams/4738/wiki/tora-gfp.png)
![](https://static.igem.wiki/teams/4738/wiki/vnp-gfp.png)

Because we were working with a vastly different design, we focused on four candidates that are representative
of each of the major secretion systems to reduce clutter instead of experimenting with each and every model.
For example, TorA was chosen as a representative group for the type II secretion system. An alternative to
the TorA secretion tag would be the GeneIII tag. Similarly, VNp6 is a chosen representative group for VNps.
There are many other variations of VNps. including VNp2 and VNp15. These decisions were made based on
extensive background research and literature review.

### Build

The same restriction digest and ligation procedures were carried out for BioBrick Assembly. The constructed
parts included a non secretion system, HlyA system, TorA system, and VNp6 system. The plasmids were then
inserted into our BL21 (DE3) E. Coli via basic transformation.

### Test

Our groups were seen to successfully fluoresce. By comparing our experimental groups with a negative control
group of untransformed E. Coli, we were able to qualitatively observe GFP-tagged phasin production in each
of our groups. However, we ran into an unexpected outcome when the non secretion system seemed to be
producing a relatively strong glow in the supernatant.

Through centrifigation, we isolated the intracellular and extracellular components of samples of each
culture. The pelleted cells were lysed using the same procedure. Then, samples from each intracellular and
extracellular groups were collected to perform fluorescence readings on a standard 96-well plate reader. The
results allowed us to compare the fluorescence values between each of the groups. A standard fluorescent
curve using GFP allowed for the computation of the concentration values of both intracellular and
extracellular GFP-tagged phasin proteins.

The results communicated the following (see Results for more):

1. In both extracellular and intracellular samples, the negative control group had minimal fluorescence
   readings verifying the lack of GFP-tagged phasin production in untransformed E. Coli.
2. In the extracellular (supernatant) samples, the type I HlyA secretion system had the highest
   fluorescence values followed by the non secretion system, the VNp6 secretion system, and type II TorA
   secretion system.
3. In the intracellular samples, the type I HlyA system had the highest fluorescence values followed by the
   non secretion system, the VNp6 secretion system, and the type II TorA secretion system.

### Learn

Our readings presented us with fluorescent measurements for the nonsecretion group that was higher than that
of TorA and VNp6 in the extracellular media. A possible explanation for these unexpected results may be due
to the hindrance of phasin-GFP fusion protein production in the presence of a type II or VNp secretion
system in combination with unintended leakage from the intracellular components of the E. coli during our
separation procedures. Future investigations should investigate an improved separation technique.

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# Restriction Digest

#### **Materials:**

| Reagent | Amount |
| --- | --- |
| Restriction Enzyme 1 | 1 µL |
| Restriction Enzyme 2 | 1 µL |
| 10x NEBuffer 2.1 | 5 µL |
| Insert or Vector DNA | 2 µL |
| Nuclease Free Water | To 50 µL |

#### **Procedure:**

1. Label PCR tubes.
2. Add all reagents to the tube.
3. Incubate for 30 minutes at 37°C. If both enzymes are Time-Saver Qualified, only incubate for 5-15
   minutes.
4. Incubate for 20 minutes at 65°C.

# Ligation

**Materials:**

| Reagent | Amount |
| --- | --- |
| T4 Ligase Buffer | 2 µL |
| T4 DNA Ligase | 1 µL |
| Vector DNA | 4 µL |
| Insert DNA | 12 µL |
| Nuclease Free Water | To 20 µL |

**Procedure:**

1. Label PCR tubes.
2. Add all reagents to the tube.
3. Incubate for 10 minutes at room temperature.
4. Incubate for 10 minutes at 65°C.

# Gibson Assembly

**Materials:**

|  | 2-3 Fragment Assembly | 4-6 Fragment Assembly | Positive Control |
| --- | --- | --- | --- |
| Total Fragments | 0.02-0.5 pmol\* X µL | 0.2-1 pmol\* X µL | 10 µL |
| Gibson Assembly Master Mix | 10 µL | 10 µL | 10 µL |
| Deionized H20 | 10-X µL | 10-X µL | 0 |
| Total Volume | 20 µL\*\*\* | 20 µL\*\*\* | 20 µL |

**Procedure:**

* Into a clean 200ul PCR reaction tube
* Add: 5 ul of each fragment (part)
* Add: 10ul of Master Mix
* Add 10ul of dH20
* If you used 2-3 fragments, place in Thermocycler for 15 min. If you used 4 or more fragments, place
  in Thermocycler for 60 min with the appropriate cycle.

# Golden Gate Assembly

**Materials:**

| **Reagent:** | **Negative Control:** | **Assembly Reaction:** |
| --- | --- | --- |
| Destination Plasmid, 75ng/µL | 1 µL | 1 µL |
| Inserts | 75-100 ng each plasmid | 2:1 molar ratio |
| NEB Golden Gate Buffer (10X) | 2 µL | 2 µL |
| NEB Golden Gate Assembly Mix | 1 µL | 1 µL |
| Nuclease Free Water | To 20 µL | To 20 µL |

**Procedure:**

1. Label PCR tubes.
2. Add all reagents to the tube.
3. Mix gently
4. Place tubes on a thermocycler
5. Choose appropriate assembly protocol:

* For 1-4 Inserts: 37°C, 1 hr → 55°C, 5 min
* For 5-10 Inserts: (37°C, 1 min → 16°C, 1 min) x 30 → 55°C, 5 min
* For 11-20 Inserts: (37°C, 5 min → 16°C, 5 min) x 30 → 55°C, 5 min

# Transformation

**Materials:**

* E. Coli Strains: BL21 (DE3)
* DNA Plasmids of Interest
* SOC Broth

**Procedure:**

1. Thaw a tube of BL21(DE3) Competent E. coli cells on ice until the last ice crystals disappear.
2. Mix gently by carefully tapping and carefully pipette 30 µl of cells into five transformation tubes
   on ice.
3. Label all tubes.
4. Add 5 µl containing 1 pg–100 ng of the respective plasmid DNA to each cell mixture. Do not add any
   plasmid to the Negative Control tube.
5. Carefully flick each tube 4–5 times to mix cells and DNA. Do not vortex.
6. Place the mixtures on ice for 30 minutes. Do not mix.
7. Heat shock at exactly 42°C for exactly 10 seconds. Do not mix.
8. Place on ice for 5 minutes. Do not mix.
9. Pipette 950 µl of room temperature SOC into each mixture.
10. Place at 37°C for 60 minutes. Shake vigorously or rotate (~5:12).
11. Transfer 100 µl of each dilution into separate labeled tubes with appropriate antibiotic and
    incubate overnight at 37°C.

**Additional Notes:**

* Thawing:
* Incubation of DNA with Cells on Ice:
* Outgrowth:
* Plating:

# 3% Glucose Media Recipe (Calgary 2017)

**Materials:**

* SOC
* Glucose
* Appropriate antibiotic if required (chloramphenicol, ampicillin)
* dH2O
* 1500-mL Erlenmeyer flask
* Stir bar
* Aluminum foil
* Hot water bath
* Overnight culture of PHB-producing bacteria

**Procedure:**

1. Make Luria-Bertani broth and after it has been autoclaved add 9g of glucose to it.
2. Microwave for 30 seconds.
3. Incubate in hot water bath at 80°C for 1-2 hours.
4. Add antibiotic to the broth (if required).
5. Inoculate the broth with 1mL of overnight culture and incubate at 37°C overnight.

# Protein Miniprep using Nickel-Based Spin Columns

**Materials:**

* Lysis/Binding Buffer
* Wash Buffer
* Elution Buffer
* NEBExpress® Ni Spin Column
* Bacterial culture (sample)

**Procedure:**

1. **Buffer Preparation**

| Buffer | 2X IMAC Buffer | 2M Imidazole | H2O | Total |
| --- | --- | --- | --- | --- |
| Lysis/Binding Buffer | 7.5 mL | 0.025 mL | 7.5 mL | 15.0 mL |
| Wash Buffer | 5.0 mL | 1.25 mL | 5.0 mL | 10.0 mL |
| Elution Buffer | 2.5 mL | 5.0 mL | 1.25 mL | 5.0 mL |

2. **Sample Preparation**

* Harvest cells by centrifugation at 4,000 x g for 15 minutes, store the pellet at -20°C or
  process immediately. Store supernatant as the extracellular component.
* Resuspend cell pellet in Lysis Buffer and lyse using the method of choice (use approximately
  5 ml of lysis buffer per gram of cell paste).
* **Note:** Cells can be lysed by standard methods, including sonication,
  repeated freeze-thaw cycles, French press, etc. Other commercially available lysis reagents
  can also be used, following manufacturer’s instructions. It is recommended that imidazole be
  omitted from any lysis buffer.
* Centrifuge the sample at 12,000 x g for 15 minutes to pellet cellular debris. Remove the
  clarified protein lysate supernatant and transfer to a new microcentrifuge tube on ice.
  Insoluble cell components will be pelleted at the bottom of the tube.
* **Note:** A standard isolation typically employs 0.5 ml of clarified lysate.
* Collect supernatant containing soluble 6xHis-tagged proteins and transfer into a fresh tube.

3. **Column Preparation**

* Remove the bottom tab of the column by twisting, loosen the top cap, and place the column in
  the collection tube provided.
* Centrifuge the column at 800 x g for 1 minute to remove the storage buffer; discard the
  buffer.
* Add 250 μl of Lysis/Binding buffer to the column.
* Centrifuge the column at 800 x g for 1 minute; discard the Lysis/Binding buffer.
* Place the column in a new 2 ml microcentrifuge tube.

4. **Lysate Binding**

* Add up to 500 μl of the protein sample lysate to the column.
* **Note:** If the sample volume is greater than 500 μl, multiple applications
  can be performed; collect the flow through in separate microcentrifuge tubes.
* Tap the column to mix the lysate with the resin and allow binding for 3 minutes.
* **Note:** Binding of some His-tagged proteins can be increased with longer
  mixing times. Cap the column and seal the bottom using the plug provided. Mix end-over-end
  at 4°C for the desired time (typically 5-15 minutes). Prolonged mixing may result in more
  non-specific binding.
* Centrifuge the column at 800 x g for 1 minute; reserve the flow through.
* Place the column in a new 2 ml microcentrifuge tube.

5. **Column Wash**

* Add 250 μl of Wash Buffer to the column and centrifuge at 800 x g for 1 minute.
* Repeat the wash step twice more, collect each wash in the same 2 ml microcentrifuge tube.

6. **Protein Elution**

* Place the column in the same 2 ml microcentrifuge tube.
* Add 200 μl of Elution Buffer to the column. Mix the resin with the elution buffer
  thoroughly.
* **Note:** Elution volume can be reduced to 100 μl if a more concentrated
  protein sample is desired; the total target protein yield may be lower.
* Centrifuge at 800 x g for 1 minute; save the eluted sample.
* Place the column in a new 2 ml microcentrifuge tube and repeat the elution step. Typically,
  >90% of the bound protein is eluted following the second elution.
* Analyze the clarified cell lysate (load), flow through, washes, and eluates by SDS-PAGE.

# Cell Lysis

**Materials:**

* Buffer NPI-10
* Lysozyme
* Benzonase®

**Procedure:**

1. Store the pellets at –20°C or at –70°C for at least 1 h.
2. Place frozen bacterial cells at room temperature and allow them to thaw for 15 min.
3. Add 100 µL buffer NPI-10 and 10 µL lysozyme solution (10 mg/ml) to the thawed cells. In addition, add 3
   units of Benzonase for every ml of the original cell culture volume (for example, for a 100 ml cell
   culture, add 300 units of Benzonase).
4. Resuspend the pellet by pipetting up and down.
5. Incubate for 30 min at either 4°C or room temperature (15–25°C).

# SDS Page Analysis

**Materials:**

* Electrophoresis Kit
* 1x SDS Glycine Buffer
* Page gel
* Aluminum foil
* Loading Dye

**Procedure:**

1. Set up the electrophoresis chamber by adding 400ml of 1x SDS - Glycine Buffer.
2. Unwrap a pre-made 15 well PAGE gel and place it in the buffer solution.
3. Carefully remove the comb and clear wells.
4. Using a hot plate or microwave, heat a beaker of water until it boils.
5. Cover with aluminum foil.
6. Tightly cap sample tubes. Push tubes through foil to suspend in the boiling water.
7. Incubate samples for 5 minutes.
8. Immediately proceed to loading the gel while the samples are still warm.
9. Connect the electrical leads to the power supply.
10. Set the voltage of the power supply and perform electrophoresis. Allow the proteins to separate on the
    gel for the recommended length of time, or until the tracking dye reaches the bottom of the gel.

# Bradford Assay

**Materials:**

* 96 well plate reader
* 1x BSA Standard
* PBS
* Coomassie Blue G-250 (Bradford Dye)
* Sample with an unknown concentration of phasin

**Procedure:**

1. Fill lane A1 with 200μL of 1x BSA standard.
2. Fill lanes A2-A12 with 100μL of PBS.
3. Transfer 100μL of A1 into A2. Pipette up and down 3 times to mix, and using the same pipette, transfer
   100μL of A2 into A3. Repeat up until A12, and after mixing A11, do not transfer to A12.
4. Pipette 20μL of the unknown sample into each individual well of the 96-well plate.
5. Add 40μL of Bradford dye to each well containing standard and sample.
6. Add 80μL of PBS to all unknown sample wells, bringing the final volume to 140μL.
7. Read absorbance at 595 nm without any prior incubation.

# GFP Fluorescence Assay

**Materials:**

* 96 well plate reader
* Control Cell Extract Containing GFP
* 1x TE Buffer
* Sample with an unknown concentration of protein

**Procedure:**

1. Fill lane A1 with 200μL of GFP Standard.
2. Fill lanes A2-A12 with 100μL of TE buffer.
3. Transfer 100μL of A1 into A2. Pipette up and down 3 times to mix, and using the same pipette, transfer
   100μL of A2 into A3. Repeat up until A12, and after mixing A11, do not transfer to A12.
4. Pipette 50μL of the unknown sample into each individual well of the 96-well plate.
5. Add 50μL of TE to all unknown sample wells, bringing the final volume to 100μL.
6. Read fluorescence at Blue 475 nm excitation (500-550 nm emission) without any prior incubation.

# Sudan Black B Solution

**Materials:**

* Sudan Black B powder
* Propylene glycol

**Procedure:**

1. Add 0.7g of Sudan Black B powder into 100 mL of propylene glycol.
2. Heat to 100~110ºC and stir until fully dissolved.

# Sudan Black B Staining for Microscopy

**Materials:**

* Bacteria Sample (in our case, E. Coli treated to produce PHB)
* Sudan Black B solution (0.3mg of solid Sudan Black B mixed with 100ml of 70% ethanol)

**Procedure:**

1. Staining will occur directly on petri dishes.
2. Directly smear alcoholic solution of Sudan Black (0.3%) on Petri dishes.
3. Incubate at 37 degrees C for 30 minutes.
4. Transfer one swab of bacteria from the petri dish to a microscope slide.
5. Swipe the slide over a Bunsen burner about 3 times.
6. Wash the stain with 96% ethanol, letting 96% ethanol run over the slide.
7. Observe with the naked eye if blue-black stains appear.
8. Move blue-black stained bacteria to slides.
9. Examine blue-black stained bacteria (PHB producers) under a microscope.

# Sudan Black B Absorbance Assay

**Materials:**

* 96 well plate reader
* Sudan Black B (SBB) solution (0.7% w/v) in propylene glycol
* Distilled water
* Sample with an unknown concentration of protein

**Procedure:**

1. Fill lane A1 with 200μL of SBB solution.
2. Fill lanes A2-A12 with 100μL of dH2O.
3. Transfer 100μL of A1 into A2. Pipette up and down 3 times to mix and using the same pipette, transfer
   100μL of A2 into A3. Repeat up until A12, and after mixing A11, do not transfer to A12.
4. Pipette SBB solution into unknown samples at a 1:10 ratio by volume of SBB solution to sample.
5. Mix either by slow centrifugation or pipetting up and down, and let sit for 40 minutes to stain.
6. Pipette 50μL of the unknown sample into each individual well of the 96-well plate.
7. Add 50μL of dH2O to all unknown sample wells, bringing the final volume to 100μL.
8. Read absorbance at 595nm without any prior incubation.

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# Phasin Secretion

After several iterations and modifications to our experimental designs, we utilized a GFP marker on our phasin proteins to detect intracellular and extracellular concentrations through fluorescence reading on a 96 well plate reader. It is important to note that we chose not to interpret the exact quantifications of concentrations between our intracellular and extracellular results due to the differences in procedural dilutions. For example, the extracellular components were directed separated and sampled after centrifugation. Meanwhile, the intracellular components were processed via cell lysis first, having been diluted at different amounts. Instead, we have compared these values in relation to each other through comparisons.

### The results communicated the following:

1. In both extracellular and intracellular samples, the negative control group had minimal fluorescence readings verifying the lack of GFP-tagged phasin production in untransformed E. Coli.
2. In the extracellular (supernatant) samples, the type I HlyA secretion system had the highest fluorescence values followed by the non secretion system, the VNp6 secretion system, and type II TorA secretion system.
3. In the intracellular samples, the type I HlyA system had the highest fluorescence values followed by the non secretion system, the VNp6 secretion system, and the type II TorA secretion system.

![](https://static.igem.wiki/teams/4738/wiki/screen-shot-2023-10-13-at-0-48-58.png)
![](https://static.igem.wiki/teams/4738/wiki/screen-shot-2023-10-13-at-0-51-49.png)
![](https://static.igem.wiki/teams/4738/wiki/screen-shot-2023-10-13-at-0-52-23.png)
![](https://static.igem.wiki/teams/4738/wiki/screen-shot-2023-10-13-at-0-52-48.png)

Our readings presented us with fluorescent measurements for the nonsecretion group that was higher than that of TorA and VNp6 in the extracellular media. A possible explanation for these unexpected results may be due to the hindrance of phasin-GFP fusion protein production in the presence of a type II or VNp secretion system in combination with unintended leakage from the intracellular components of the E. coli during our separation procedures. Future investigations should investigate an improved separation technique.

# PHB Production

Before moving on to designing our PHB secretion system, we wanted to confirm our selected from model for PHB production in E. coli. Our system is based on part Part:BBa\_K934001 from Tokyo Tech 2012. The team used gas chromatography and mass spectrometry (GC/ MS) in order to identify the products. Other past teams have also utilized chloroform in order to attain PHB. However, with our limited resources, we found an alternative to these methods by verifying PHB production through staining and microscopy.

We followed a simple Black Sudan B staining protocol to prepare slides of our experimental PHB-producing E. coli cells and untransformed E. coli as our negative control for comparison.

![](https://static.igem.wiki/teams/4738/wiki/screen-shot-2023-10-13-at-0-54-45.png)

# PHB Secretion

Following the success of our PHB production model, we moved forward by integrated PHB production with the earlier secretion models (more information can be found in Engineering Success). After incubating our E. coli cells for PHB production in a 3% glucose media, we quantified the extracellular and intracellular concentrations of PHB through absorbance readings on the 96 well plate reader. Additionally, we verified these results through a second fluorescence reading to confirm a correlation between phasin and PHB production with concentrations of GFP.

The results communicated the following (see Results for more)

1. In both intracellular and extracellular samples, the negative control group had minimal fluorescence readings verifying the absence of GFP-tagged phasin production.
2. In the extracellular (supernatant) samples, the VNp6 system had the highest fluorescence values followed by the non secretion system, the HlyA system, the TorA system, and the negative control. The non secretion system had the highest absorbance values followed by the negative control, the HlyA system, the TorA system, and the VNp6 system.
3. In the intracellular samples, the VNp6 system had the highest fluorescence values, followed by the non secretion system, the HlyA system, the TorA system, and the negative control. The negative control and HlyA had the highest absorbance values, the non-secretion system, the VNp6 system, and the TorA system.

![](https://static.igem.wiki/teams/4738/wiki/screen-shot-2023-10-13-at-0-58-29.png)
![](https://static.igem.wiki/teams/4738/wiki/screen-shot-2023-10-13-at-0-59-06.png)

We realized our chosen method of PHA quantification is restricted in accuracy and reliability. For future reference, in order to optimize the accuracy of PHA determinations with spectrophotometric quantification, we learnt that a calibration curve comparing known PHA values and measured absorbance values of Sudan Black B dilutions is a necessary component for quantifying PHA values. We suggest following the improved methodology proposed by Porras et al. However, if possible, we recommend future teams use techniques such as HPLC, gas chromatography, or flow cytrometry to obtain results.

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