iGEM 2023 — Team leiden

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
{% block page\_content %}

# {{ self.title() }}

Overview of all experiments performed to optimize PHA production using *M. extorquens*

* ## Content
* [![](https://static.igem.wiki/teams/4721/wiki/hp/cirkels-24.svg)
  Introduction](#Introduction)
* [![](https://static.igem.wiki/teams/4721/wiki/hp/cirkels-24.svg)
  Promoter characterization](#slide1-chapter1)
* [![](https://static.igem.wiki/teams/4721/wiki/hp/cirkels-24.svg)
  Overexpression of *phaC*, *groEL* and *groES*](#slide1-chapter2)
* [![](https://static.igem.wiki/teams/4721/wiki/hp/cirkels-24.svg)
  Carotenoid biosynthesis pathway knockout](#slide1-chapter3)
* [![](https://static.igem.wiki/teams/4721/wiki/hp/cirkels-24.svg)
  Inducible autolysis system](#slide1-chapter4)
* [![](https://static.igem.wiki/teams/4721/wiki/hp/cirkels-24.svg)
  PHA extraction and *M. extorquens* cultivation](#slide1-chapter5)
* [![](https://static.igem.wiki/teams/4721/wiki/hp/cirkels-24.svg)
  PHA identification](#slide1-chapter6)
* [![](https://static.igem.wiki/teams/4721/wiki/hp/cirkels-23.svg)
  Back to the top](#top)

# Introduction

Below you can find the lab notes of all the performed experiments in chronological order. They are grouped per research line:

**1.** Promoter characterization

**2.** Overexpression of *phaC*, *groEL* and *groES*

**3.** Carotenoid biosynthesis pathway knockout

**4.** Inducible autolysis system

**5.** PHA extraction and *M. extorquens* cultivation

**6.** PHA identification

The description and results of each research line can be found on the
[Engineering page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D)
and
[Results page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D), respectively.
The detailed protocols can be found on the
[Protocols page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D). The notebook was written using Benchling.

##### **1.** Promoter characterization

# Notebook

[Raw data](https://doi.org/10.5281/zenodo.8420742)

##### **2.** Overexpression of *phaC*, *groEL* and *groES*

# Notebook

[Raw data](https://doi.org/10.5281/zenodo.8422542)

##### **3.** Carotenoid biosynthesis pathway knockout

# Notebook

##### **4.** Inducible autolysis system

# Notebook

[Raw data experiment 1](https://doi.org/10.5281/zenodo.8420881)

[Raw data experiment 2](https://doi.org/10.5281/zenodo.8420816)

##### **5.** PHA extraction and *M. extorquens* cultivation

# Notebook

[Raw data](https://doi.org/10.5281/zenodo.8423134)

##### **6.** PHA identification

# Notebook

[Raw data fluorescence microscopy](https://doi.org/10.5281/zenodo.8420587)

[Raw data MALDI-TOF](https://doi.org/10.5281/zenodo.8420645)

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{% extends "layout.html" %}
{% block title %}Engineering{% endblock %}
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# {{ self.title() }}

Improving PHA production of *M. extorquens* AM1 by
iterative optimization cycles

## Cycle A

Characterizing various promoters for use in genetic engineering of *M.
extorquens*.

## Cycle B

Overexpression of genes for improved PHA production.

## Cycle C

Enhancing PHA production by alleviating metabolic burden through gene knockout.

## Cycle D

Finding the optimal cell lysis system for PHA extraction.

* ### Content
* [![](https://static.igem.wiki/teams/4721/wiki/hp/cirkels-24.svg)
  Engineering success](#engineering-success)
* [![](https://static.igem.wiki/teams/4721/wiki/hp/cirkels-24.svg)
  Cycle A](#Cycle-A)
* [![](https://static.igem.wiki/teams/4721/wiki/hp/cirkels-24.svg)
  Cycle B](#Cycle-B)
* [![](https://static.igem.wiki/teams/4721/wiki/hp/cirkels-24.svg)
  Cycle C](#Cycle-C)
* [![](https://static.igem.wiki/teams/4721/wiki/hp/cirkels-24.svg)
  Cycle D](#Cycle-D)
* [![](https://static.igem.wiki/teams/4721/wiki/hp/cirkels-24.svg)
  Conclusion](#Conclusion)
* [![](https://static.igem.wiki/teams/4721/wiki/hp/cirkels-24.svg)
  Future experiments](#Future)
* [![](https://static.igem.wiki/teams/4721/wiki/hp/cirkels-22.svg)
  References](#References)
* [![](https://static.igem.wiki/teams/4721/wiki/hp/cirkels-23.svg)
  Back to the top](#top)

# Engineering success

With PHAse Out, our goal was to make PHA production cost-effective and
sustainable. We wanted to improve PHA production in *Methylobacterium
extorquens (M. extorquens)* AM1, which is able to use methanol as its carbon
source to make PHA. See our
[sustainability page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27sustainable%27%29%20%7D%7D) to read more about this process.

To improve PHA production, we employed the Design-Build-Test-Learn (DBTL) cycle
(Fig. 1). This engineering cycle is an important framework for synthetic
biology, with each cycle consisting of various elements that collectively build
the final system. Going through each cycle allows an iterative and thorough
process.

Firstly, we reviewed existing literature and developed an initial project
design. Secondly, as can be read on our
[Human Practices page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D) we interviewed experts and implemented their feedback into our project design.
We applied the DBTL cycle in various aspects of our project. On this page, we
explain how we used the cycles to engineer our research lines. The interaction
between our four engineering cycles can be seen in Figure 2.

![DBTL cycle](https://static.igem.wiki/teams/4721/wiki/engineering/engineering-cycle-06-1.png)

**Fig. 1 |** The Design-Build-Test-Learn (DBTL) Cycle

![DBTL cycles](https://static.igem.wiki/teams/4721/wiki/engineering/engineering-cycle-05-1.png)

**Fig. 2 |** Overview of the 4 engineering cycles and how they interact. Cycle A:
Promoter. Cycle B: Over-expression. Cycle C: Knockout. Cycle D: Autolysis.

# Cycle A

## Characterizing promoters for optimal induction and overexpression systems in *M. extorquens*

The genetic engineering tools available for *M. extorquens* are not
comparable with those available for model organisms like *Escherichia
coli*. Hence, Cycle A focused on characterization of various constitutive
and inducible promoters to determine their potential for application in Cycle B
and Cycle D, which required constitutive and inducible promoters, respectively.

## Design & Build

We constructed plasmids consisting of pTE100 backbone with a promoter and
*mCherry* reporter gene. Constitutive and inducible promoters were chosen
to incorporate in further cycles.

## Test & Learn

Promoters were tested by measuring mCherry fluorescence and OD600.
Best promoters were *P*mxaF (constitutive) and PL/O4/A1
(inducible). These were incorporated in later cycles.

## Redesign

Future experiments would focus on retesting promoters and expanding the amount
of promoters available for *M. extorquens*.

## Design & Build

With regard to the *phaC* and *groES/EL* overexpression cycle
(Cycle B), various constitutive promoters were assessed. The promoter with the
highest known expression in *M. extorquens* is the *mxaF*promoter1. *P*mxaF is the promoter for the *mxaF*
gene in *M. extorquens*. This gene encodes the large subunit of methanol
dehydrogenase1, which is highly expressed in methanol-rich
conditions2, it has also been reported to be slightly upregulated on
methanol3 and since we grew our cells on a methanol-based medium, we
considered the expression of this promoter constitutive.

Since high levels of overexpression is not ideal in each situation, we also
wanted to test promoters with lower expression rates than *mxaF*.
[Dr. Lennart Schada von Borzyskowski](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D) published a paper on
several promoters that were characterized in *M. extorquens* that we
identified as possible alternatives to the *mxaF* promoter1.
These promoters include the promoters to the fumarase coding *fumC*
gene*,* the cytochrome c oxidase subunit II coding *coxB* gene and
the transcription elongation factor thermo-unstable *tuf* gene. These
promoters are referred to as *P*fumC (5-15%), *P*coxB (40-50%) and
*P*tuf (70-100%) with increasing promoter strength relative to
*P*mxaF (100%)1. All three promoters are considered
constitutive.

In terms of the autolysis system, we wanted to assess different inducible
promoters. We chose to clone different inducible promoters that were hardly
characterized for *M. extorquens*. We looked into the IPTG inducible
promoters PA1/O4/O3 and PL/O4/A14, cumate
inducible promoters PQ5  and PQ21485 and
vanillate inducible promoter PV106 and and OHC14
inducible promoter pCinR7, 8. As can be seen in Figure 3, the
inducible promoters have a similar structure, where a repressor gene is
constitutively expressed, which binds to the operator sequence. When the ligand
is present, the repressor releases, and mCherry is expressed. For
PL/O4/A1, for example, the *lacI* repressor gene is
constitutively expressed by the PlacIq promoter, and binds to the
lacO operator sequence inside the PL/O4/A1 promoter region, thereby
repressing the promoter. When the ligand IPTG is present, this binds LacI,
releasing it from the operator. Then, PL/O4/A1 is open for
expression, leading to gene expression of the reporter gene mCherry. In general,
repression is not complete, meaning there will always be some level of
expression. The level of uninduced expression is indicated as the ‘leakiness’ of
the promoter. In this cycle, we wanted to know how leaky the inducible promoters
were, since we wanted to express a lethal gene construct, only at a desired
time, so that it is able to grow normally before then.

A schematic overview of the mCherry testing cassette with different promoters is
shown in Figure 3. An overview of all the characterized promoters is elaborated
on in the
[Parts page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27parts%27%29%20%7D%7D).

![Promoter construct](https://static.igem.wiki/teams/4721/wiki/engineering/promoter-construct.png)

**Fig. 3 |** **A:** Schematic overview of the mCherry testing cassette
with different constitutive promoters. **B:** Schematic overview of the mCherry testing cassette with different inducible
promoters.

As a backbone, we used pTE100 empty vector1 containing an
oriV-traJ’ origin with TcR. To clone the promoters and testing
cassettes in the backbone, we used restriction cloning to insert the promoters,
and Gibson cloning to insert the fluorescent reporter gene mCherry into the
constructs. We were not able to construct, transform and test each plasmid given
limited time. Some plasmids were available to us with either the promoter or the
promoter and mCherry already. The *P*mxaF, *P*fumC, *P*coxB
and *P*tuf plasmids had both the promoter and mCherry,
PL/O4/A1, PA1/O4/03 and pCinR only needed to have mCherry
put in. All plasmids were constructed and transformed into *M.
extorquens*. However, due to time constraints we were unable to test
PQ2148 and PA1/O4/03.

## Test & Learn

We wanted to learn which of the constitutive promoters were most optimal for the
*phaC* overexpression and which inducible promoters were most optimal for
the autolysis system. Therefore we
[measured](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D)the mCherry
fluorescence signal of the successfully cloned strains with a plate reader
(Tecan Plate reader Infinite 200 PRO). Using the fluorescence signal and the
cell density, the dose-dependent promoter strength was determined. You can find
all the results on the
[Results page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D).

![Promoter activity](https://static.igem.wiki/teams/4721/wiki/engineering/engineeringbarplot-data.png)

**Fig. 4 | Promoter activity** of the four constitutive promoters,
and of the inducible promoters both in uninduced (-) and induced (+) conditions.
The control was a strain transformed with a plasmid carrying mCherry without a
promoter. The dotted line serves as a visual aid and represents the fluorescence
signal of 250 nM of the Sulforhomadine from the iGEM calibration kit, as part of
the
[Interlab study](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27interlab%27%29%20%7D%7D). For PL/O4/A1 a 190% increase in
expression was seen when induced compared to non induced condition (Student
t-test, p< 0.0001). A 30% increased expression was seen for PV10 when
grown under induced conditions in the first replicate the promoter was tested
(Student t-test, p< 0.05). For the other inducible promoters, barely any
fluorescence was seen, as well as no effect of the inducer.

Out of the constitutive promoters, *P*mxaF showed the most fluorescence
compared to control (Fig. 4). Since we did not know what level of overexpression
of *phaC* and *groEL/ES* would be beneficial for PHA production, and
at what point the overexpression would start to burden the cells, we decided to
use all the constitutive promoters for the *phaC* and *groEL/ES*overexpression.

For the inducible promoters, the results were not as promising as expected (Fig.
4). By mistake, the pCinR strains were induced with IPTG instead of OHC14, which
explains the lack of induced effect seen, which is why this data is omitted. The
PV10 strain was first tested with freshly prepared vanillate stock,
and an increase in mCherry signal was seen, and later, parallel strains were
tested as well with these same dilutions. The absence of induction could be
explained by the use of not freshly prepared inducers, which is why on the
[Results page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) the results of this replicate are omitted. However,
to solidify conclusions about the characteristics of this promoter, the
experiments need to be repeated. The PQ5 promoter showed surprisingly
little fluorescence. We omitted this part from our list since the signal was
even lower than the negative control, indicating a technical problem for this
measurement. The PL/O4/A1 promoter showed the highest effect by the
presence of the inducer IPTG. Since this promoter was also already characterized
in literature4 and we found a strong effect of the inducer, we used
this promoter for induced autolysis in Cycle D.

## Redesign

For continuation of this cycle, we would redo the experiment for the strains we
were not able to analyze yet. We also want to retest the strains mentioned here
to solidify our conclusions and expand the parts collection of promoters for
*M. extorquens*. Moreover, in the redesign of this experimental line, we
would like to test even more promoters, such as this newly published variant of
the cumate-inducible promoter PQ21489.

This would provide us with more knowledge and would expand the genetic
engineering tools that are available for *M. extorquens.* For example, we
made plasmids expressing the mCherry under cumate inducible promoters, but were
not able to get good data on these strains. In the redesign of this experiment,
we would like to test this again, integrating the knowledge from this cycle to
be able to gather more data on this inducible promoter. Since IPTG is
relatively expensive, it would be beneficial if an inducible system that has a
cheaper and more sustainable ligand as inducer, such as cumate, could be used
for large scale production. For example, we characterized the cumate inducible
promoter with this in mind, as cumate is 10 times cheaper than IPTG, with cumate
costing €52.10 per 5 gram and IPTG costing €492.00 per 5 gram10. On
an industrial scale, this would allow for significant savings.

# Cycle B

## Increasing PHA production through overexpression of native genes in *M. extorquens*

The metabolism of wildtype (WT) *M. extorquens* is not optimized for
large scale production of PHA11. A powerful tool for enhancing the
production of a compound such as PHA is the overexpression of native
genes12. Using our constitutive promoters from Cycle A, we designed a
construct to constitutively express endogenous synthase genes to increase
production levels of PHA.

## Design & Build

Constructs were created consisting of a constitutive promoter tested in cycle A
and either *phaC*, *groEL/ES* or *phaC* and
*groEL/ES* genes combined.

## Test & Learn

PHA production was quantified by Nile Red staining. For each gene the optimal
promoter was different, but all showed an increase compared to wildtype.

## Redesign

In future experiments co-overexpression of the genes under different promoters
could increase PHA production, as well as combining these constructs with the
carotenoid knockout strain (Cycle C).

## Design & Build

Consulting with
[dr. Schada von Borzyskowski](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D), we decided
to overexpress the native *phaC*, *groEL* and *groES*
genes. PhaCserves as the synthase for PHA, while the GroEL/ES complex
is a chaperone involved in efficient folding of PhaC13. This
complexalso prevents the formation of aggregates and is hypothesized
to increase PHA production13.

Based on the results of the characterization from Cycle A, we made various
genetic constructs. These constructs are visualized in Fig. 5. Overall, we
created constructs with four different promoters, with either *phaC, groEL/ES*or *phaC* and *groEL/ES* genes combined. In addition, we
included a version with a histidine rich region (HIS tag) for the extraction of
the protein.

![phac construct](https://static.igem.wiki/teams/4721/wiki/engineering/promoter-phac.png)

**Fig. 5 | Visualization of the different genetic constructs for overexpression**
with four different promoters *P*mxaF, *P*fumC, *P*coxB and *P*tuf, with either
*phaC, groEL/ES* or *phaC* and *groEL/ES* genes combined.

## Test & Learn

We **tested** the constructs by growing the bacterial strains in
liquid culture and quantifying PHA production with Nile Red (NR) staining
according to
[experiment 2:](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) Overexpression of genes for increased
PHA production allowed us to compare the amount of PHA produced by our strains
to the WT.

The results of the PHA quantification of the different strains are summarized in
Fig. 6. All constructs show increased PHA production in comparison to the WT at
48 hours. However, there was no promoter that was consistently better across all
constructs. We **learned** that the optimal promoterdiffers between the different endogenous genes tested. The data is further
elaborated on the
[Results page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D).

![phac graphs](https://static.igem.wiki/teams/4721/wiki/engineering/layout-3a-engineering.png)

**Fig. 5 |** Graphs showing the PHA production of the
different strains. **A.** PHA production PhaC overexpression. **B.** PHA production GroEL/ES overexpression.
**C.** PHA production PhaC and GroEL/ES overexpression.

PHA production at 48 hours of growth, supplemented with 0.5% methanol at 44
hours, where the promoters are shown on the x-axis and the PHA production in
mg/mL is shown on the y-axis.

## Redesign

In this cycle, we have learned that overexpression of *phaC* and
*groEL/ES* genes together resulted in increased PHA production in
comparison to the wildtype, where overexpression of phaC resulted in the biggest
increase.

Based on these findings, in future experiments we could construct separate
plasmids for the *groEL/ES* genes and the *phaC* gene, under the
expression of *P*tuf and *P*mxafrespectively. We
hypothesize that co-overexpression under different promoters would improve the
synergistic effects. Furthermore, we want to combine the highest PHA producing
overexpression strains with the carotenoid knockout strain of Cycle C. This
could be achieved through transformation of the Δ*crtI* strain with the
overexpression plasmid. Theoretically, this could lead to a *M.
extorquens* strain that allows for a higher influx of compounds into the PHB
cycle and overexpression for even more production of PHA. The final strain would
then be optimized according to all we learned in regard to the promoters,
overexpression and the knockout in cycles A, B and C.

# Cycle C

## Enhancing PHA production of *M. extorquens* by knocking out a competing pathway to optimize the metabolic flux

To increase PHA production, we investigated the possibilities of changing the
metabolic flux in favor of PHA production. This could be achieved by knocking
out specific pathways that are superfluous for a production strain. In *M.
extorquens,* a promising target for this is the carotenoid biosynthesis
pathway14. This pathway creates pigments for UV protection that are
not relevant in a microbial bioproduction setting, and can therefore be knocked
out15.

## Design & Build

A metabolic model of *M. extorquens* was used to identify gene targets
for a carotenoid pathway knockout. Constructs were created to knock out these
genes using homologous recombination.

## Test & Learn

The *crtl* gene was successfully knocked out. The knockout of the *ispA*
and *dxr* gene were unsuccessful and PHA quantification was not possible
due to time constraints.

## Redesign

Gibson assembly could be used to overcome some of the difficulties with cloning. After
testing, the carotenoid knockout could be combined with the PHA overexpression
strain (Cycle B).

## Design & Build

To optimize metabolic engineering, we employed a genome-scale metabolic
[model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D) of *M. extorquens*, identifying key knockout targets and
assessing their impact on carotenoid biosynthesis. We predicted an increased
carbon flux to the PHA production pathway (PHB cycle) to improve PHA production,
see Figure 7. Three promising gene targets for a knockout were selected:
*dxr*, *ispA*, and *crtI*. This was supported by literature
where a colorless *M. extorquens* *crtI* knockout strain had been
found to be viable14.

![metabolism](https://static.igem.wiki/teams/4721/wiki/engineering/metabolism-pink.png)

**Fig. 7 |** Visualization of the effect of the carotenoid biosynthesis knockout
on PHA production.

Various genetic tools, such as CRISPR-Cas based technologies, are available for
gene deletion, but are not always efficient in non-model organisms, such as
*M. extorquens*16. Therefore, we opted for a method that has
been established for the use in alphaproteobacteria, including *M.
extorquens*, which relies on two homologous recombination (HR) events
between the genome and a non-replicating plasmid (pREDSIX) bearing DNA regions
that surround the gene of interest17.

A kanamycin resistance (KmR) gene was isolated from a versatile antibiotic
resistance gene donor plasmid (pRGD-KmR) and inserted in between the flanking
regions, 750 base pairs each, which will only be integrated into the genome and
be active upon homologous recombination. If only a single crossover takes place,
the full plasmid is integrated into the genome and the target gene is not
replaced. However, this mutant will have an active antibiotic resistance gene,
so the antibiotic selection is not sufficient to discriminate between a single
or double crossover mutant. To be able to specifically select colonies with
successful target gene replacement, pREDSIX contains mCherry, which will be
active if a single crossover occurs, leading to the expression of a fluorescent
protein. Antibiotic selection and non-fluorescence thus enable the selection of
correct mutant strains. The genetic construct can be seen in Figure 8.

See our
[Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) and
[Notebook](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D) page for details on
the cloning of these constructs.

![metabolism](https://static.igem.wiki/teams/4721/wiki/engineering/carotenoid-gene-construct.png)

**Fig. 8 |** pREDSIX plasmid with flanking regions for the target genes. KmR:
Kanamycin resistance cassette. AmpR: ampicillin resistance cassette.
FrUp: flanking region upstream. FrDown: flanking region
downstream. Ori: origin of replication.

## Test & Learn

Based on our validation criteria, we concluded that the *crtI* gene was
successfully knocked out. However, knocking out the *ispA* gene and the
*dxr* gene repeatedly failed. For the *ispA* gene this was due to
cloning failure, because of time constraints we were not able to test a
different type of cloning. We were not able to obtain a viable *dxr* knockout
despite numerous transformation attempts. This may indicate
that *dxr* is an essential gene, and that this gene deletion is lethal, which is supported by
literature18. A more detailed overview of our results can be found on
the
[Results page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D): Knockout of genes for increased PHA
production. We aimed to quantify the amount of PHA that our *crtI*knockout strain produced, however, due to time constraints this was not
possible. This would have been performed in the same manner as described in
Cycle B.

We were not able to test the system, but our hypothesis was that by alleviating
the metabolic load there would be more energy available for the PHB cycle,
resulting in more PHA production. Furthermore, our
[computational predictions](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D) suggested that growing the knockout in nitrogen-deficient
conditions may further increase PHA production. As such, validation of the
strain and model would have also included growing the knockout in these
conditions.

During our experiments, we faced challenges in gene knockouts within *M.
extorquens* and the carotenoid biosynthesis pathway. The phenotype of
our *crtI* knockout strain also did not match expectations,
[remaining pink](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D) despite literature reporting colorless mutants with
*crtI* knockouts14. Moreover, since the restriction cloning of
the *ispA* knockout continually failed, Gibson assembly could be an
alternative route for obtaining this construct in future experiments.

## Redesign

In this cycle, we have hypothesized that a knockout in the carotenoid
biosynthesis pathway creates a change in the metabolic flux in favor of PHA
production.To redesign this cycle, we would incorporate our learnings from both
Cycle B (overexpression) and C (knockout). Drawing from the overexpression
cycle, we would integrate the best-performing strains into the viable carotenoid
knockout strain, creating an optimized *M. extorquens* strain for PHA
production. Simulating the carotenoid biosynthesis pathway gene knockouts in the
metabolic network model [Modeling page] of *M. extorquens* showed that
the reaction flux through the PHB cycle is increased compared to the wild-type,
except for the reaction synthesized by the PhaC enzyme. This highlights the need
for combining a carotenoid knockout strain with a *phaC* overexpression
strain to overcome this potential limitation.

# Cycle D

## Uncovering the optimal inducible autolysis system for *M. extorquens*

Current methods for PHA extraction are environmentally hazardous, costly, and
dangerous for workers19, 20. To address this, we used cell lysis
systems from bacteriophages. Phages are viruses that infect bacteria, and due to
this, they have cell lysis systems for killing and escaping their host
cells21. If these lysis systems can be expressed in *M.
extorquens*, at the desired time, this could benefit the extraction
processes.

## Design & Build

5 lysis genes were selected from phages and codon optimized for *M. extorquens*.
These genes were tested together with an inducible promoter.

## Test & Learn

No difference between induced and uninduced conditions was visible.
Gp110 endolysin and EJ1
Holin/Endolysin showed the highest lethality.

## Redesign

In future experiments, a different inducible promoter should be tested. The
optimal promoter should show no leakiness and have an inexpensive inducer.

## Design & Build

The optimal choice for a phage-based cell lysis system would be a phage specific
to *M. extorquens*, however, these have not been found yet. Therefore, we
selected 5 lysis genes and gene systems from phages that infect bacteria that
are phylogenetically closely related to *M. extorquens*. See Table 1 on
the
[Results page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) for an overview of the lysis genes and their
host organisms.

Due to the highly lethal nature of these genes, we used the inducible promoter
PL/O4/A1, as characterized in Cycle A. This was done to allow
selective activity of the lethal genes.

As exogenous DNA is not always functional in a new host organism, we also
optimized several aspects of our genetic constructs to ensure their
functionality in *M. extorquens* using the De Novo DNA transcription
level prediction software and the De Novo DNA combined synthesis success tool
and codon optimization tool22. The genetic constructs can be seen in
Figure 9.

![construct autolysis](https://static.igem.wiki/teams/4721/wiki/engineering/copy-of-promoter-doodles-1.png)

**Fig. 9 | Structure of the autolysis gene cassettes.** Including
inducible promoter PL/O4/A1 and RBS from pET-16B*,* followed
by the following phage-derived lysis genes: *Escherichia coli* phage T4
lysozyme, *Gallus gallus* lysozyme, *Agrobacterium. Tumefaciens*
phage T7 hydrolase, *Salmonella* Bacteriophage Gp110 endolysin, and
*Pneumococcus* phage EJ1 Holin/Endolysin. Description of the
phage-derived genes can be found in Table 1 on the
Structure of the autolysis gene cassettes. Including
inducible promoter PL/O4/A1 and RBS from pET-16B*,* followed
by the following phage-derived lysis genes: *Escherichia coli* phage T4
lysozyme, *Gallus gallus* lysozyme, *Agrobacterium. Tumefaciens*
phage T7 hydrolase, *Salmonella* Bacteriophage Gp110 endolysin, and
*Pneumococcus* phage EJ1 Holin/Endolysin. Description of the
phage-derived genes can be found in Table 1 on the results pages [results page]
[Results page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D).

See our
[Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) and
[Notebook](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D) pages for details on the
cloning of these constructs.

## Test & Learn

Firstly, we tested the systems by comparing the effect of the gene inserts on
colony forming unit frequency in both the induced and uninduced state, as can be
seen in fig. 10. This showed a statistically significant decrease in the number
of surviving colony forming units for all cell lysis systems. The reduction was
especially notable for *Salmonella* phage endolysin Gp110 (GP110) and
*Pneumococcus* EJ1 Holin/Endolysin (EJ1), with these showing high
lethality. However, there was no difference between the induced and uninduced
conditions, indicating that the genes were functional, but the inducer did not
work.

![graph autolysis](https://static.igem.wiki/teams/4721/wiki/engineering/results-survival-autolysis.png)

**Fig. 10 |** Survival study set-up for assessing autolysis systems under inducing
and non inducing conditions in *M. extorquens,* compared to positive (+)
and negative (-) controls. Biological duplicates employed, with confidence
intervals given for calculated population variance.

Secondly, to confirm the effects of the inserted genes, we studied the effect of
the genes on the cell wall integrity of *M. extorquens* cells. This was
done by growing controls and cell lines under exposure to butanol, an organic
solvent. *M. extorquens* is generally highly resistant to solvent
toxicity, and this trait is closely linked to cell wall structure and
integrity23. If the lysis genes had a cell wall compromising effect,
this would be noticeable under solvent stress. Both Gp110 and EJ1 showed a
significantly decreased tolerance to butanol in comparison to the other strains
and to the control. This supports our hypothesis that these inserts compromise
the cell wall of *M. extorquens*. See
[Results page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) for more
information. Based on this, we could conclude with a good degree of certainty
that two of the phage lysis gene constructs were functional in *M.
extorquens*.

However, the lysis system did not work entirely as anticipated. Our results
indicate that the PL/O4/A1 inducer was either too leaky, or did not
repress our specific genes well. Despite this, all lysis systems showed
statistically significant effects in either the survival study, the solvent
exposure experiment, or both.

## Redesign

This cycle shows that the PL/O4/A1 promoter is not fully repressed in
the absence of IPTG. We found that especially Gp110 endolysin and EJ1
Holin/Endolysin show statistically significant lethality.

As found in Cycle A, the IPTG inducible promoter PL/O4/A1 had a high
level of leakiness, and to iterate on the design of this cycle, we would need to
employ an alternative inducer system. Due to the leakiness, we were unable to
characterize the effect of the auto-lysis system on extraction efficiency.
Future iterations for the auto-lysis system should include screening multiple
inducers to identify an optimally non-leaky and affordable inducible system for
large-scale industrial use.

# Conclusion

Our engineering cycles allowed for optimization of PHA production of *M.
extorquens*, eventually leading to improved yields and cost-efficiency. The
cycles combine data analysis, modeling, experimentation, and iterative
refinement to achieve our goal. Going through the DBTL cycles allowed us to
obtain results, negative and positive, learning from them and then adjusting the
process. Despite not being able to complete every cycle due to time constraints,
each cycle did help us obtain important results that could be used in other
cycles. Overall, all of the experiments allowed us to gain more knowledge about
how to produce cost-effective and sustainable PHA.

# Future experiments

As the engineering cycle is an iterative process, it is never fully completed.
Since an iGEM project only lasts a few months, there is a limited amount of
experiments that can be performed. Therefore, there are various experiments and
steps that PHAse Out can take in the future. These include assessing the
reproducibility of the results obtained here, and then incorporating all of the
learnings from the different cycles to engineer an optimized *M. extorquens*strain that includes overexpression, knock-out, and autolysis.

##### References

1. Schada Von Borzyskowski L, Remus-Emsermann M, Weishaupt R, Vorholt
   JA, Erb TJ. A set of versatile brick vectors and promoters for the assembly,
   expression, and integration of synthetic operons in methylobacterium extorquens
   am1 and other alphaproteobacteria. *ACS Synth Biol*. 2015;4(4):430-443.
   doi:10.1021/sb500221v
2. Liu Q, Kirchhoff JR, Faehnle CR, Viola RE, Hudson RA. A rapid method for
   the purification of methanol dehydrogenase from Methylobacterium extorquens.
   *Protein Expr Purif*. 2006;46(2):316-320. doi:10.1016/J.PEP.2005.07.014
3. Carrillo M, Wagner M, Petit F, Dransfeld A, Becker A, Erb TJ.
   Design and Control of Extrachromosomal Elements in Methylorubrum extorquens AM1.
   *ACS Synth Biol*. 2019;8(11):2451-2456. doi:10.1021/acssynbio.9b00220
4. Marx CJ, Lidstrom ME. Development of improved versatile
   broad-host-range vectors for use in methylotrophs and other Gram-negative
   bacteria. *Microbiology (N Y)*. 2001;147:2065-2075.
5. Kaczmarczyk A, Vorholt JA, Francez-Charlot A. Cumate-inducible gene
   expression system for sphingomonads and other Alphaproteobacteria. *Appl
   Environ Microbiol*. 2013;79(21):6795-6802. doi:10.1128/AEM.02296-13
6. Kaczmarczyk A, Vorholt JA, Francez-Charlot A. Synthetic
   vanillate-regulated promoter for graded gene expression in Sphingomonas.
   Published online 2014. doi:10.1038/srep06453
7. Meyer AJ, Segall-Shapiro TH, Glassey E, Zhang J, Voigt CA.
   Escherichia coli “Marionette” strains with 12 highly optimized small-molecule
   sensors. *Nat Chem Biol*. 2019;15(2):196-204.
   doi:10.1038/s41589-018-0168-3
8. Schuster LA, Reisch CR. A plasmid toolbox for controlled gene expression
   across the Proteobacteria. *Nucleic Acids Res*.
   2021;49(12):7189-7202.doi:10.1093/nar/gkab496
9. Pöschel L, Gehr E, Jordan P, Sonntag F, Buchhaupt M. Expression of
   toxic genes in Methylorubrum extorquens with a tightly repressed,
   cumate-inducible promoter. *Antonie van Leeuwenhoek, International Journal of
   General and Molecular Microbiology*. Published online September 26,
   2023:1-10. doi:10.1007/S10482-023-01880-7/FIGURES/5
10. Yuan S, Zhang R, Cao Y, Guo J, Xian M, Liu W. New expression
    system to increase the yield of phloroglucinol. *Biotechnology &
    Biotechnological Equipment*. 2020;34(1):405-412.
    doi:10.1080/13102818.2020.1764386
11. Suzuki T, Yamane T, Shimizu S. Kinetics and effect of nitrogen
    source feeding on production of poly-β-hydroxybutyric acid by fed-batch culture.
    *Appl Microbiol Biotechnol*. 1986;24(5):366-369.
    doi:10.1007/BF00294591/METRICS
12. Borrero-de Acuña JM, Poblete-Castro I. Rational engineering of
    natural polyhydroxyalkanoates producing microorganisms for improved synthesis
    and recovery. *Microb Biotechnol*. 2023;16(2):262.
    doi:10.1111/1751-7915.14109
13. Goto S, Miyahara Y, Taguchi S, Tsuge T, Hiroe A. Enhanced Production
    of (R)-3-Hydroxybutyrate Oligomers by Coexpression of Molecular Chaperones in
    Recombinant Escherichia coli Harboring a Polyhydroxyalkanoate Synthase Derived
    from Bacillus cereus YB-4. *Microorganisms*. 2022;10(2).
    doi:10.3390/MICROORGANISMS10020458/S1
14. Van Dien SJ, Marx CJ, O’Brien BN, Lidstrom ME. Genetic
    Characterization of the Carotenoid Biosynthetic Pathway in Methylobacterium
    extorquens AM1 and Isolation of a Colorless Mutant. *Appl Environ
    Microbiol*. 2003;69(12):7563. doi:10.1128/AEM.69.12.7563-7566.2003
15. Mo XH, Sun YM, Bi YX, et al. Characterization of C30 carotenoid
    and identification of its biosynthetic gene cluster in Methylobacterium
    extorquens AM1. *Synth Syst Biotechnol*. 2023;8(3):527. doi:10.1016/J.SYNBIO.2023.08.002
16. Volke DC, Orsi E, Nikel PI. Emergent CRISPR–Cas-based technologies
    for engineering non-model bacteria. *Curr Opin Microbiol*.
    2023;75:102353. doi:10.1016/J.MIB.2023.102353
17. Ledermann R, Strebel S, Kampik C, Fischer HM. Versatile vectors for
    efficient mutagenesis of Bradyrhizobium diazoefficiens and other
    alphaproteobacteria. *Appl Environ Microbiol*. 2016;82(9):2791-2799.
    doi:10.1128/AEM.04085-15/SUPPL\_FILE/ZAM999117112SO1.PDF
18. Mo XH, Zhang H, Wang TM, et al. Establishment of CRISPR
    interference in Methylorubrum extorquens and application of rapidly mining a new
    phytoene desaturase involved in carotenoid biosynthesis. *Appl Microbiol
    Biotechnol*. 2020;104(10):4515-4532.
    doi:10.1007/S00253-020-10543-W/FIGURES/7
19. Favaro L, Basaglia M, Casella S. Improving polyhydroxyalkanoate
    production from inexpensive carbon sources by genetic approaches: a review.
    *Biofuels, Bioproducts and Biorefining*. 2019;13(1):208-227.
    doi:10.1002/BBB.1944
20. Yabueng N, Napathorn SC. Toward non-toxic and simple recovery
    process of poly(3-hydroxybutyrate) using the green solvent 1,3-dioxolane.
    *Process Biochemistry*. 2018;69:197-207.
    doi:10.1016/J.PROCBIO.2018.02.02
21. Young RY. Bacteriophage lysis: mechanism and regulation.
    *Microbiol Rev*. 1992;56(3):430. doi:10.1128/MR.56.3.430-481.1992
22. De Novo DNA: The Future of Genetic Systems Design and Engineering.
    Accessed October 4, 2023. https://www.denovodna.com/software/
23. Belkhelfa S, Roche D, Dubois I, et al. Continuous culture
    adaptation of methylobacterium extorquens AM1 and TK 0001 to very high methanol
    concentrations. *Front Microbiol*. 2019;10(JUN).
    doi:10.3389/FMICB.2019.01313/FULL

{% endblock %}


{% extends "layout.html" %} {% block title %}Results{% endblock %}
{% block page\_content %}

# {{ self.title() }}

An overview of our results

## Characterization of promoters for *M. extorquens*

Characterizing promoters for regulating gene expression in *M.
extorquens* for genetic engineering.

## Overexpression of genes for increased PHA production

Overexpressing PHA synthase and chaperones to increase PHA production.

## Knock out of genes for increased PHA production

Knocking out the carotenoid pathway to increase available metabolic carbon.

## Introduction of heterologous genes for autolysis

Making and testing inducible self lysis systems for *M. extorquens*.

* ### Content
* [![](https://static.igem.wiki/teams/4721/wiki/hp/cirkels-24.svg)
  Introduction](#slide1-chapter1)
* [![](https://static.igem.wiki/teams/4721/wiki/hp/cirkels-24.svg)
  Promoter characterization](#slide1-chapter3)
* [![](https://static.igem.wiki/teams/4721/wiki/hp/cirkels-24.svg)
  Gene overexpression](#slide1-chapter4)
* [![](https://static.igem.wiki/teams/4721/wiki/hp/cirkels-24.svg)
  Gene knockout](#slide1-chapter5)
* [![](https://static.igem.wiki/teams/4721/wiki/hp/cirkels-24.svg)
  Autolysis system](#slide1-chapter6)
* [![](https://static.igem.wiki/teams/4721/wiki/hp/cirkels-24.svg)
  Conclusion](#slide1-chapter7)
* [![](https://static.igem.wiki/teams/4721/wiki/hp/cirkels-22.svg)
  References](#slide1-chapter8)
* [![](https://static.igem.wiki/teams/4721/wiki/hp/cirkels-23.svg)
  Back to the top](#top)

# Introduction

> ## **Our non-model organism**
>
> In our experiments, we work with the strain *Methylobacterium extorquens*
> AM1Δ*cel1*, a pink-pigmented facultative methylotrophic
> α-proteobacterium2. The Δ*cel* mutation ensures *M.
> extorquens* does not form biofilms, which is advantageous in an experimental
> setting since it ensures reliable measurements of its growth curves1.
> From here on, this strain is referred to as *M. extorquens* wild type.
> *M. extorquens* can utilize methanol as the sole carbon source and
> convert this into polyhydroxyalkanoates (PHA). *M. extorquens* has been
> proposed as a promising methanol-based bioproduction platform3.

The overarching goal of PHAse Out was to make cost-effective and sustainable PHA
with green methanol as a feedstock. For this, we genetically engineered
*Methylobacterium extorquens* AM1 with the aim of increasing PHA
production and improving PHA extraction methods. On this page, we show the most
important results that substantiate our progress towards this goal.

![Overview](https://static.igem.wiki/teams/4721/wiki/project-description/expertimental-overview-final2.png)

**Fig. 1 |**  **Research lines of PHAse Out.** Visual
overview of the experimental lines, their interconnectivity, and connection to
the project aim.

# Research lines

## Characterization of promoters for *M. extorquens*

|  |  |
| --- | --- |
| Goal | Characterize various inducible and constitutive promoters for genetic engineering of *M. extorquens* |
| Method | Cloning of the promoters into the empty pTE100 backbone with the *mCherry* reporter gene. Transformation into *M. extorquens*. Measure the mCherry fluorescence signal over time under different inducer concentrations |
| Hypothesis | The cells with constitutive promoters are expected to show higher fluorescence compared to the control having no promoter. The cells with inducible promoters are expected to show dose-dependent increase in fluorescence levels in relation to the inducer |
| Conclusion | The IPTG- and vanillate-inducible promoters showed significantly increased fluorescence when induced, where the induction of the IPTG inducible promoter PL/O4/A1 was the strongest. Out of the constitutive promoters, *P*mxaF had the highest expression, followed by *P*tuf. *P*fumC and *P*coxB showed similarly low expressions |

To effectively modify *M. extorquens*, we required genetic elements that
were proven to be functional in the organism. The toolkit for genetic elements
of this organism is lacking when compared to standardized model organisms4,
5. We first created a collection of parts containing different promoters
from literature, all of which are described in more detail on the
[Parts page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27parts%27%29%20%7D%7D). We characterized multiple inducible and constitutively active
promoters to use in the *phaC* overexpression and autolysis experiments.
We tested the constitutive promoters [*P*mxaF](http://parts.igem.org/Part%3ABBa_K4721002),
[*P*fumC](http://parts.igem.org/Part%3ABBa_K4721000), [*P*coxB](http://parts.igem.org/Part%3ABBa_K4721001)
and [*P*tuf](http://parts.igem.org/Part%3ABBa_K4721003)4,
Isopropyl β-d-1-thiogalactopyranoside [(IPTG) inducible promoter](http://parts.igem.org/Part%3ABBa_K4721006) PL/O4/A15 and
[vanillate inducible promoter](http://parts.igem.org/Part%3ABBa_K4721008)PV106. To learn how we constructed
and transformed the different plasmids4, please visit the [Protocols](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D) and [Notebook pages](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D).

We used a *M. extorquens* optimized mCherry reporter cassette to assess
promoter strength. As a negative control, a strain carrying a plasmid containing
the mCherry testing cassette, without a promoter, was used. As a positive
control plasmid developed by dr. Schada von Borzyskowski were used4.
For the promoters that were tested, the inducible promoter sequences were cloned
in front of the mCherry cassette. A growth experiment was set up with biological
and technical duplicates, where the OD600nm and fluorescence were
tracked.

In Figure 2, the fluorescence data indicating the promoter strength is shown as
the mCherry fluorescence normalized to the OD600nm. The sampling time
was placed at 24 hours of growth, which fell around the middle of the
exponential phase of growth. This is further elaborated on in the dropdown below
Figure 2. For PL/O4/A1, the 48-hour time point was instead used since
the growth curve showed a delay. Contrary to our expectation, one of the
biological duplicates of *P*coxB did not show fluorescence. This
indicates an aberration suppressing mCherry expression in this specific
biological duplicate, therefore the data from this duplicate was excluded from
the analysis.

For the constitutive promoters, the expression levels were similar to the
promoter activities described in literature4. *P*mxaF had the
highest expression, followed by *P*tuf. *P*fumC and *P*coxB
which showed similarly low expressions. For the inducible promoters, multiple
concentrations of the respective inducers were used. In Figure 2, the uninduced
condition was compared to the highest induced condition. There was a significant
190% increase for PL/O4/A1 between the uninduced and induced
conditions and a significant 30% increase in expression for PV10. For
results with a broader range of inducer concentrations, please visit the
specific information submitted to the parts registry for each
[part](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27parts%27%29%20%7D%7D).

![Promoter overview](https://static.igem.wiki/teams/4721/wiki/parts/resultsbarplot-data.png)

**Fig. 2 |** **Normalized fluorescence indicating promoter strength** of
the four constitutive promoters, and of the inducible promoters both in
uninduced (-) and induced conditions (+). The mean of the biological and
technical duplicates is shown. The error bars represent the standard deviation.
The dotted line serves as a visual aid and represents the fluorescence signal of
250 nM of the Sulforhomadine from the iGEM calibration kit, as part of the
Interlab study. For PL/O4/A1 there was a significant increase of 190%
between the uninduced and induced conditions (*Student’s T-test*, \*\*\*\* p
< 0.0001). For PV10 there was a significant 30% increase in
expression when induced (*Student’s T-test*, \* p < 0.05 Student’s
T-test).

##### Determination of exponential phase of growth

Figure S1 shows the growth curves which were used to determine the optimal time
point to sample the fluorescence in order to determine promoter strength, which
is in the middle of the exponential phase7. In addition, the growth
curves assess the viability of the strains, thereby giving an indication of the
toxicity of the construct and potential inhibition by inducer presence. The
bacteria carrying the different mCherry expressing plasmids were expected to
have similar growth curves. However, the curves show that presence of the
*P*mxaF, *P*coxB and *P*tuf constructs inhibited the
maximum OD600nm reached when compared to the control and the
*P*fumC strain. The growth curves of the PV10 strains show
reduced growth. For PL/O4/A1 , a delayed growth was seen. Based on
these fluorescence measurements, the sampling time was set to be 24 hours for
all strains, except PL/O4/A1, which was set at 48 hours.

![Promoter overview](https://static.igem.wiki/teams/4721/wiki/results/results/od-results.png)

**Fig. S1 |** Growth curves for the mCherry expressing strains,
showing the mean of the biological and technical duplicates of each promoter,
with the dotted lines representing the error bars. A sigmoidal 4PL curve (black)
is fitted on each graph. For PL/O4/A1, the uninduced (-) and the 1000
µM IPTG induced (+) conditions are shown, and for PV10 the uninduced
(-) and the 250 µM vanillate induced (+) conditions. This graph indicates that
most strains were in their exponential phase of growth at 24h. An exception is
the strain with the PL/O4/A1, where the growth was delayed. The
middle of its exponential phase is at around 48h. PV10 shows
inhibited growth, both in the uninduced (-) as induced (+) condition.

To further expand the genetic engineering toolkit for *M. extorquens,*follow up experiments may include characterization of a larger set of
constitutive and inducible promoters.

## Overexpression of genes for increased PHA production

|  |  |
| --- | --- |
| Goal | Increase PHA production |
| Method | Overexpression of the PHA synthase gene (p*haC*) and the gene coding for the GroEL/ES chaperone complex through various constitutive promoters |
| Hypothesis | Overexpression of the *phaC* and *groEL/ES* genes will increase PHA production in recombinant strains in comparison to the wild type |
| Conclusion | Overexpression of both *phaC* and *groEL/ES* genes resulted in increased PHA production in comparison to the wild type, with overexpression of *phaC* under control of the *P*mxaF promoter resulting in a 24-fold increase of PHA production. Co-overexpression of *phaC* and *groEL/ES* under control of the *P*tuf promoter indicated possible synergistic effects with a 24-fold increase of PHA production |

To improve PHA production, we chose to overexpress the PHA synthase gene
*phaC*, which codes for Poly(3-hydroxyalkanoate) polymerase and is a key
enzyme in the PHA production pathway8. This pathway in *M.
extorquens* is further elaborated on our
[Modeling page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D). As
expression of this gene in the wild type *M. extorquens* is regulated by
available nutrients in the environment4, we aimed to express
*phaC* independent of this regulation and in high quantity.Literature suggests that overexpression of the GroES/EL chaperone system
assists in folding the produced PhaC enzyme, improving the active protein levels
of this enzyme9. Therefore, we created genetic constructs with the
constitutive promoters of variable expression strengths that were tested in
cycle A of
[engineering](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D). Both the *phaC* and*groES/EL* genes were amplified from *M. extorquens* genomic DNA with
primers designed for Gibson Assembly using the NEBuilder10.
*PhaC* and *groEL/ES* were amplified for both individual cloning
and a combination of the two in one plasmid. Figure 3 shows the created
constructs.

![phac construct](https://static.igem.wiki/teams/4721/wiki/engineering/promoter-phac.png)

**Fig. 3 |**  Genetic constructs that were created for overexpression and
co-overexpression. With four different promoters, *P*mxaF,
*P*fumC, *P*coxB and *P*tuf, with either *phaC*,
*groEL/ES* or *phaC* and *groEL/ES* genes combined.

See the experimental design in further detail on the
[Protocols page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D).

### Nile Red staining

To quantify the PHA production of our cultures, we used fluorescent Nile Red
staining, which stains PHA and biomass11,12. As Nile Red does not
exclusively stain PHA, the background signal from the biomass must be
determined. For this, a *phaC* knockout strain (Δ*phaC*, provided
by Dr. Schada von Borzyskowski from Leiden University) was used. By removal of
the *phaC* gene, the Δ*phaC* strain did not produce PHA and was
used to extrapolate the amount of PHA present in PHA producing strains. This was
done by staining both the samples with Nile Red and subtracting the Δ*phaC*fluorescence signal from the fluorescent signal of the other sample. All
samples were grown as explained in the optimal cultivation conditions segment,
with methanol replenishment at 40h post-inoculation and sample collection at 48h
post-inoculation. The collected samples were then used for biomass determination
and fluorescence measurement. The wild type strain was used here as validation
of this quantification technique for further experiments. The results are shown
below in Figure 4.

![Fluo nile red](https://static.igem.wiki/teams/4721/wiki/results/new-wt-vs-dphac-linear-range.png)

**Fig. 4 |**  Fluorescence signal from Nile Red staining of
Δ*phaC* and *M. extorquens* wild type at different biomasses, with
the biomass indicated in mg/ml. Nile Red stains biomass and PHA. As
Δ*phaC* does not produce PHA, the signal from the Δ*phaC* can be
subtracted from the signal of the PHA producing strain to quantify the amount of
PHA produced. The divergence of the wild type sample at 0.8 mg/mL, marked with an '\*' is due to a
[handling error](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D) and this point was subsequently excluded from the analysis.

The Nile Red staining of the wild type and Δ*phaC* strain show a
biomass-dependent signal increase, with the wild type showing a greater
fluorescent signal than the Δ*phaC*. This is due to PHA accumulation in
the wild type. Subtracting the fluorescent signal from the Δ*phaC* strain
allows for quantification of PHA. This can then be used to quantify PHA
accumulation via Nile Red staining in milligrams per milliliter culture.

##### Optimizing cultivation conditions to balance nutrient availability for maximum PHA accumulation

To determine optimal cultivation conditions for PHA production, three different
cultivation methods were adapted from literature13-15. These included
the addition of one of three medium components 40 hours post-inoculation, when
cells were in the
[stationary phase](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D). The exact execution of the
experiment can be read on our
[Protocols page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D).

| Cultivation conditions 40 hours post-inoculation | Hypothesis |
| --- | --- |
| Methanol (0.5% (v/v)) replenishment | The carbon source is the limiting factor for PHA production. Adding methanol resolves this15. |
| Nitrogen-free medium replenishment | Replenishment of minerals and methanol as well as a stimulus in the form of lacking nitrogen is required for PHA production13. |
| Medium replenishment | Addition of all medium components will lead to increased PHA production14. |

At different time points during cultivation, samples of equal amounts of biomass
were taken. These samples were subsequently stained with Nile Red to determine
PHA related fluorescence12 in response to different culture
conditions. The results are shown in Figure S2.

![Biomass nile red](https://static.igem.wiki/teams/4721/wiki/results/file-for-sophie.png)

**Fig. S2 |**  Triplicate samples of biomass stained with Nile Red
at 15, 18, 35, 40, 56, 62, and 79 hours post-inoculation. In the stationary
phase at 40 hours post-inoculation either the medium, methanol, or nitrogen free
medium was replenished.

Out of the three medium replenishment conditions, the results show the highest
fluorescence intensity increase for the methanol replenishment condition. The
highest fluorescence was measured at 56 hours post-inoculation. Hence, the
replenishment of methanol at 40 hours post-inoculation was chosen as the
preferred cultivation condition in further experiments. This is also
advantageous due to the speed and simplicity of this method in large scale
bioreactors, as this method does not require a complete medium change to induce
PHA production15. In subsequent experiments PHA production was
measured at 48 hours, rather than 56 hours, due to practical considerations.

To determine the intensity of the Nile Red signal from PHA we made a calibration
curve with a sample of 99% pure PHA16. Sonicated PHA was stained with
nile red and the fluorescence was measured. Read more about our calibration
experiments in our
[Protocols page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D). The calibration curve is
shown in Figure 5.

![Calibration](https://static.igem.wiki/teams/4721/wiki/results/g75.png)

**Fig. 5 |** PHA calibration curve acquired through Nile Red
staining and measurement of sonicated PHA of known concentrations. PHA samples
were prepared, stained, and measured in quintuplicate. This curve, in
combination with the measurements from Figure 4, was used in further experiments
to quantify PHA in the different *M. extorquens* strains.

### *phaC* overexpression

To test the effect of *phaC* overexpression on PHA production, we
inoculated strains with the *P*mxaF, *P*fumC, and *P*tuf
promoters and a wild type strain. The bacteria were cultured following the above
established culture conditions, where initial inoculation in minimal
methanol-based medium is followed by methanol replenishment after 40 hours.
Samples were collected at 48 hours after inoculation and were used for biomass
determination as well as Nile Red staining. Measurements were done in technical
quadruplicates. The results are shown below in Figure 6. All strains containing
an overexpression construct showed a significant increase in PHA production
compared to the wild type (p < 0.0001). In line with previously obtained
results, overexpression by *P*mxaF yielded the highest PHA production,
with a 24-fold increase. Contrary to expectation, *P*tuf’s expression
strength does not translate to PHA accumulation in this experiment.
*P*fumC-driven PHA accumulation is significantly less than that of
*P*mxaF (p = 0.0103), prompting us to use *P*mxaF for future
experiments.

![pha prod phac](https://static.igem.wiki/teams/4721/wiki/results/pha-per-strain-phac.png)

**Fig. 6 |**  PHA production of *phaC* overexpressing strains
with the different promoters in comparison to the wild type at 48 hours after
inoculation in technical quadruplicates. *P*mxaF is the highest producing
strain, with a 24-fold increase in comparison to the wild type and a significant
(Student’s T-test, \* p = 0.0103) increase in comparison to the second highest
producing strain, *P*fumC.

Following the same workflow, GroES/EL overexpressing strains with
*P*mxaF, *P*fumC, *P*coxB, and *P*tuf constructs
were cultured and measured. The results in Figure 7 show a significant increase
in all overexpressing strains compared to the wild type (p ≤ 0.0001), with a
~5-fold increase in PHA production in *P*mxaF, *P*fumC and
*P*coxB and a 10-fold increase in PHA production in *P*tuf.
*P*tuf also shows a significant production increase in comparison to
*P*coxB (p = 0.001).

![pha prod groe](https://static.igem.wiki/teams/4721/wiki/results/pha-per-strain-groe.png)

**Fig. 7 |** PHA production of the GroES/EL overexpression strains
with the different promoters in comparison to the wild type at 48 hours.
*P*tuf shows a significant increase in PHA accumulation over all other
strains (Student’s T-test, \*\* p = 0.001)

Lastly, strains co-overexpressing *phaC* and *groES/EL* under the
control of *P*fumC, *P*coxB, and *P*tuf, respectively, were
tested as described above. *P*mxaF was not tested, as cloning failed and
could not be repeated due to time constraints. Again, all strains show a
significant increase in PHA production over wild type (p < 0.0001), with a
10-fold increase for *P*fumC, a 11-fold increase in *P*coxB and a
24-fold increase in *P*tuf, as shown in Figure 8. The increase in PHA
production through co-overexpression of *phaC* and *groES/EL*
under *P*tuf is similar to the increase through overexpression of
*phaC* under *P*mxaF. Under *P*tuf, a synergistic effect
was observed for the co-expression constructs in comparison to the single
constructs. The PHA production of the co-expression construct surpasses that of
the single protein expressing strains. Especially promising is the fact that
co-overexpression of the two enzymes with *P*tuf results in a PHA
production within 3% of the PHA production of the *P*mxaF driven
*phaC* overexpression strain. This also represents a ~5-fold increase
from sole *phaC* overexpression by *P*tuf. Future experiments
could investigate whether the same effect can be observed with *P*mxaF,
yielding an even stronger PHA-producing strain, due to the inherently stronger
promoter. *P*fumC does not indicate a similarly potential synergistic
effect, but rather an antagonistic one. Under *P*fumC the results
obtained for the co-expression construct showed lower PHA production than for
sole *phaC* overexpression.

![pha prod phacgroe](https://static.igem.wiki/teams/4721/wiki/results/phac-groe.png)

**Fig. 8 |** PHA production of the *phaC* and *groES/EL*
overexpression combination strains with the different promoters in
comparison to the wild type at 48 hours after inoculation.

In conclusion, overexpression, and co-overexpression of *phaC* and
*groES/EL* show promising results for increased PHA production and
possible synergistic effects. The results from our preliminary studies could be
further supplemented through protein extraction and enzyme activity assays.

Negative promoter protein interactions in *Methylobacterium spp.* have
previously been reported with fluorescent reporter proteins. Investigating
whether this is the case with our constructs would be necessary for explaining
differences between promoter strengths from validated mCherry-based testing to
*groES/EL* overexpression.

### Microscopy verification of PHA in overexpression strains

We visualized PHA accumulation via the staining of granules of PHA located in
*M. extorquens*. To substantiate this design choice and verify the
presence of PHA in our overexpression lines, we carried out the Nile red
staining protocol on the Δ*phaC*, wild type, *phaC*
overexpression, *groES/EL* overexpression, and *phaC + groES/EL*
overexpression lines under the control of *P*mxaF (*phaC + groES/EL*under *P*fumC). We then imaged these cells under a fluorescence
microscope and compared them (Figure 9) For more details, see the Fluorescence
Microscopy
[Protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D).

![pha prod phacgroe](https://static.igem.wiki/teams/4721/wiki/results/pha-microscopy.png)

**Fig. 9 |**  Comparison of *M. extorquens* Δ*phaC*,
wild type, *phaC* overexpression, *groES/EL* overexpression, and
*phaC + groES/EL* overexpression lines after staining, 535nm excitation,
605nm emission12.

In these images, there are several promising indications. Δ*phaC* is
devoid of granules, while the wild type had small bodies located at the cell
poles which responded positively to staining, indicating they are lipophilic and
therefore likely PHA. The +*phaC* stain showed similar results, except
with larger, brighter bodies, not always located at the poles. groE consistently
showed smaller, but more numerous vesicles spread across the length of the cell.
The combination *phaC + groES/EL* did not show an increase over these,
being mostly similar to the +*phaC*. From this we can conclude that there
is a cellular basis for the quantification of PHA levels via Nile Red staining,
and that the overexpression and chaperones had an effect, however no obviously
superior overexpression was visible at the cell level.

## Knockout of carotenoid genes for increased PHA production | | | | --- | --- | | Goal | Knock out genes in the carotenoid biosynthesis pathway to increase flux of acetyl-coa into PHA production | | Method | Replace genes encoding for enzymes involved in the carotenoid biosynthesis pathway by a kanamycin resistance gene | | Hypothesis | Gene targets were selected based on computational simulations using a metabolic network model of *M. extorquens*, which were predicted to be most effective in improving PHA production | | Conclusion | A Δ*crtI* strain was successfully created and validated | To improve the production of PHA by *M. extorquens*, we analyzed the [metabolic network model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D) to identify promising gene knockout targets that guide the metabolic flux towards the PHB cycle. Based on the computationally predicted results, three target genes encoding enzymes involved in the carotenoid pathway were selected: *dxr*, *ispA* and *crtI* (Figure 10). ![knockout](https://static.igem.wiki/teams/4721/wiki/results/results-knockout-targets.png) **Fig. 10 |** Overview of selected gene knockout targets *dxr*, *ispA* and *crtI* involved in the carotenoid biosynthesis pathway, which are predicted to effectively eliminate carotenoid production and enhance PHA production in *M. extorquens,* as based on our modeling results. A detailed explanation of the construct design can be found on the [Engineering page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D). The upstream and downstream flanking regions for homologous recombination of *crtI*, *dxr* and *ispA* were successfully inserted into the pREDSIX plasmid and validated by [sequencing](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D). The subsequent insertion of the kanamycin resistance gene was successful for the vectors containing the flanking regions of *crtI* and *dxr*. Unfortunately, the insertion of the kanamycin resistance cassette into the vector containing the flanking regions of *ispA* proved to be difficult and no verified vector could be obtained during the lab time. Transformation of pREDSIX containing the flanking regions of *crtI* and the kanamycin resistance gene into *M. extorquens* was successful and non-fluorescent colonies were identified using fluorescence microscopy. Five non-fluorescent colonies were further used to validate the knockout strain. The genomes of these five colonies were isolated and the gene replacement was validated by amplifying the target region in the genome which should have been replaced by a kanamycin resistance gene in the transformed strains. As the kanamycin resistance cassette is roughly the same size as the native *crtI* gene, the amplified DNA was subsequently digested using NdeI, which should only digest the target sequence containing the kanamycin resistance cassette and not the sequence containing the *crtI* gene. The expected bands from a correct Δ*crtI* mutant are 1228, 964 and 948 bp, compared to 3433 bp for wild type *M. extorquens*. The results can be seen in Figure 10 and correspond to the expected band lengths. ![knockout](https://static.igem.wiki/teams/4721/wiki/results/dcrti-validation-gel.png) **Fig. 11 |** Validation of Δ*crtI* colonies (1-5) by amplifying the target region in the genome. Amplified DNA was digested using NdeI Expected bands of a correct Δ*crtI* mutant are 1228, 964 and 948 bp, compared to 3433 bp for wild type *M. extorquens*. Correct bands are highlighted in red. Interestingly, the obtained Δ*crtI* strains seemed as pink as wild type *M. extorquens*, contrary to previous research that showed that deletion of *crtI* resulted in a colorless *M. extorquens* mutant17. Due to time limitations, the PHA production by these Δ*crtI* mutants could not be quantified. This would have been performed similar to the PHA quantification of the *phaC* overexpression strain using NR staining and subsequently validated with extraction. Furthermore, the transformation of pREDSIX containing the flanking regions of *dxr* and the kanamycin resistance gene into *M. extorquens* repeatedly failed. This may indicate that *dxr* is an essential gene, and that this gene deletion is lethal, which has also been suggested previously18.

## Introduction of heterologous genes for autolysis

### 1. Determining the effectiveness and induction of exogenous Holins/Endolysins

|  |  |
| --- | --- |
| Goal | Introduce plasmids containing holin/endolysin genes to assess whether *M. extorquens* is able to express and use these proteins to auto-lyse |
| Method | Introduce the five synthetic IPTG-induced holin/endolysin plasmids, and test the lethality of induced and uninduced genes by colony survival counts |
| Hypothesis | Induced genes should show a reduction in colony counts compared to the uninduced, with the most lethal gene having the lowest surviving colony count |
| Conclusion | PL/O4/A1 is not fully repressed in the absence of IPTG, however all genes show statistically significant lethality, especially Gp110 endolysin and EJ1 Holin/Endolysin |

To improve the efficiency of PHA extraction and remove the need for hazardous
chemicals to lyse the cells, we were interested in using inducible cell lysis
systems to cause cell death on demand. We selected 5 cell lysis genes that had
been experimentally verified in related bacteria and codon optimized them for
expression in *M. extorquens*. We inserted native ribosome binding sites
(RBS) from pTE-16B, which is an established plasmid in *M. extorquens,*
before all protein coding sequences, ensuring effective transcription by the
host's RNA polymerase.

The high GC content of *M. extorquens* and phage DNA (60%+) is
problematic to synthesize. The standard codon table of *M. extorquens*
also differs from *E. coli*. To combat this, the *De Novo DNA*
combined synthesis success tool19 was used to strike a balance
between accurate amino acid codon sequences and successful synthesis
probability. The synthesized genes can be seen below (Table 1).

**Table 1 | Self lysis systems, origin, brief description and literature source.**

| Name | Origin | Description | Source |
| --- | --- | --- | --- |
| T4 Lysozyme | *Escherichia coli* phage T4 | Fusion protein of a membrane lysis protein with cell penetrating peptide | 20 |
| Gg Lysozyme | *Gallus gallus* (chicken), modified for efficiency in *P. putida* | *Gallus gallus* (chicken) lysozyme sequence | 21 |
| T7 hydrolase | *Agrobacterium Tumefaciens* phage T7 | Modified *A. tumefaciens* phage lytic system, peptidoglycan hydrolase | 22 |
| Gp110 endolysin | *Salmonella* Bacteriophage | *Salmonella* Bacteriophage peptidoglycan hydrolase | 23 |
| EJ1 Holin/Endolysin | *Pneumococcus* phage EJ1 | Two part Holin-Endolysin system derived from *Pneumococcus* phage EJ-1 | 24 |

To transform the genes into *M. extorquens* and selectively
induce them, we used the inducible plasmid pTE100-PL/O4/A1. We used
Gibson assembly cloning to create the constructs. We verified the functionality
of the genes by sequencing the inserted regions. Experiments were continued with
sequencing-verified plasmids. See the
[Protocols page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D) for detailed
information about the construct cloning and transformation into *M.
extorquens*.

Since we expected a lethal phenotype for induced holin/endolysin systems, we
used a survival study setup to assess the effect of the inserts in *M.
extorquens*. We used pTE100-PL/O4/A1 without insert as our
negative control, as it is expected to be non-lethal, and a plasmid free
competent *M. extorquens* culture as our positive control, as this should
be totally lethal due to lacking antibiotic resistances. The details for this
can be found on the
[Protocols page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D).

![survival](https://static.igem.wiki/teams/4721/wiki/engineering/results-survival-autolysis.png)

**Fig. 13 |** Survival study for assessing autolysis systems under
inducing and non-inducing conditions in *M. extorquens,* compared to
positive (non-lethal) and negative (lethal) controls. Biological duplicates
employed, with confidence intervals calculated from mean and variance of the
duplicates.

These results were statistically analyzed (two tailed T-test, p < 0.05) by
comparing the uninduced average colony counts with the average colony count for
the controls, then comparing the induced and uninduced populations of the same
gene. This showed that all the autolysis systems have statistically decreased
populations in comparison to the negative control (non-lethal genotype). The
Gp110 endolysin and EJ1 Holin/Endolysin showed the most significant decrease in
population, going as low as single digit colony counts. Between the IPTG
negative and IPTG positive plates, there was no significant difference in colony
counts. This combination indicates that the autolysis cassettes influenced
colony survival, however, autolysis cassettes showed similar effects on the
population under uninduced and induced conditions. From this it can be concluded
that the inducer was “leaky” and was not sufficiently repressed under
non-induced conditions, allowing for expression of lysis genes even when no
inducer was present.

### 2. Determining the effects of Holins/Endolysins on solvent sensitivity

|  |  |
| --- | --- |
| Goal | Test the effect of the heterologous Holins/Endolysins on the solvent resistance of *M. extorquens* |
| Method | Using the transformed *M. extorquens* from the survival experiment, spot plating on a butanol-containing plate and compare colony growth and opacity |
| Hypothesis | Colonies carrying genes with lower colony forming unit averages will have lower opacity colonies compared to wild type |
| Conclusion | High lethality genes show reduced growth on Butanol plates |

The previous results indicate a correlation between reduction in cell viability
and the autolytic systems. However, this correlation cannot be fully
substantiated to determine causation. As such, we performed further analysis of
the effect of the autolysis systems on cell wall integrity. Solvent tolerant
bacteria, such as *M. extorquens,* are reliant on the integrity of their
cell walls, efflux pumps, and various other mechanisms for their resistance to
solvents25. *M. extorquens* is generally solvent tolerant due
to its methylotrophic nature. Given that the lysis genes should negatively
affect cell well integrity, we examined the difference in solvent tolerance
between our engineered strains and a wild type control by exposing them to
butanol. See
[Protocols page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D) for more information on the
experimental set-up.

![survival rates](https://static.igem.wiki/teams/4721/wiki/results/layout-butanol-experiment.png)

**Fig. 12 |**  Survival rates of *M.
extorquens* cells carrying autolysis genes on varying butanol
concentrations, IPTG induced and uninduced states. Survival rates quantified via
image derived grayscale values. Experiments were performed in technical
triplicates.

Statistical analysis of the rate of change in growth in response to butanol
shows a significant difference (p < 0.05) between Gp110 endolysin and EJ1
Holin/Endolysin as compared to the other strains, with Gp110 and EJ1 showing
less cell viability than the other strains. Additionally, there is a significant
difference (p < 0.05) between the Gp110 endolysin and EJ1 Holin/Endolysin and
the control, with these displaying less cell viability than the control. The
Gp110 endolysin and EJ1 Holin/Endolysin strains show a near zero cell viability
under solvent stress compared to the control and other cell lines. This supports
our previous results, which indicate the Gp110 endolysin and EJ1 Holin/Endolysin
inserts have the largest effect on cell viability. As the inserted genes lyse
cells by either hydrolyzing the cell wall, by inserting holin proteins into the
cell membrane, or through a combination of these mechanisms, the solvent
experiment results support our hypothesis that the insertion of these genes
into *M. extorquens* reduced cell wall integrity. Taken together, the
experiments performed for the development of an autolysis system in *M.
extorquens* show the functional incorporation of the inserts, a significant
negative effect on cell viability, and a significant effect of Gp110 endolysin
and EJ1 Holin/Endolysin on solvent tolerance.

In conclusion, we were able to successfully insert and verify the presence of
our autolysis genes. After this, we indicated all autolysis genes reduced the
number of colony forming units after transformation and growth on plates, and
two of these genes were causing significantly reduced cell viability under
solvent stress. We were unable to detect significant increases in cell death
after induction with IPTG. The combination of both a reduced colony forming unit
rate and a reduced tolerance to solvents in our most lethal genes supports the
cause of cell death being related to the integrity of the cell membrane of the
*M. extorquens* cells*.* The target of our autolysis genes were
the cell wall and membrane, therefore this supports that we were able to
successfully induce autolysis in *M. extorquens.* However, as we were not
able to control the induction of the autolysis system, we were unable to test
the viability of this process for increased PHA extraction
efficiency.

##### PHA extraction

Solvent extraction using chloroform is the most well established extraction
method for PHA26. Extraction expert
[Jonathan Salt](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D) recommended using solvent-based extraction with chloroform and
acetone, since it is cheapest for large scale extraction methods. However, due
to concerns raised by
[safety experts](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D), we focussed
on DMC-based solvent extraction27. Read more about the safety
concerns of using chloroform on our
[Safety page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27safety%27%29%20%7D%7D). The complete protocol
can be found on our
[Protocols page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D). No discernible product quantities
were extracted, and no increase in extracted product compared to the
Δ*PhaC* strain
[was found](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D).

To test whether the fault laid with the extraction or if the strains did not
produce PHA, a solvent extraction using chloroform was performed. 572 milligram
of extracted product was obtained from 150mL of wild type *M. extorquens*
(Figure S3). Due to time constraints, optimizing the DMC-based extraction was
not possible. It was likewise not possible to analyze the product from the
chloroform extraction. To improve DMC-based extraction in future experiments we
recommended using a dry-pellet for extraction, as the adhesiveness of the wet
cell pellet to the falcon tubes was difficult to work with when transferring the
wet-pellet with DMC to the extraction set-up.

![PHA](https://static.igem.wiki/teams/4721/wiki/results/picture-chloroform-extraction.png)

**Fig. S3 |**  Obtained product from a chloroform-based extraction,
extracted from 150mL of wild type *M. extorquens*

## Conclusion

Through our experimental workflow, PHAse Out aimed to establish
a cost-effective and sustainable method for producing PHA with
*M. extorquens* using green methanol. Our main achievements and
findings include the successful characterization of various
promoters, notably demonstrating significant increases in
fluorescence for IPTG- and vanillate-inducible promoters,
with the IPTG-inducible promoter PL/O4/A1 standing out.
Among the constitutive promoters, *P*mxaF exhibited the
highest expression. Furthermore, overexpression of the PHA synthase gene (*phaC*)
and the GroEL/ES chaperone complex resulted in a remarkable
24-fold increase in PHA production, especially when controlled
by the *P*mxaF promoter. Co-overexpression of *phaC* and *groEL/ES*
under the *P*tuf promoter also indicated possibly promising synergistic effects. The development and integration of autolysis systems with exogenous
holin/endolysin genes showed a significant reduction in cell viability,
with Gp110 endolysin and EJ1 Holin/Endolysin displaying the most
pronounced effects on cell integrity. These systems have potential
applications in cell lysis processes. In addition, the assessment of solvent tolerance revealed that specific
autolysis genes significantly reduced cell viability under solvent stress,
confirming their influence on cell wall integrity. With these findings, we further and support the scientific developments
made toward creating sustainable PHA production methods, with the ultimate
goal of creating a more sustainable future.

##### References

1. Delaney NF, Kaczmarek ME, Ward LM, Swanson PK, Lee M-C, Marx CJ. Development
   of an optimized medium, strain and high-throughput culturing methods for
   Methylobacterium extorquens. *PLoS One*. 2013;8(4):e62957.
   doi:10.1371/journal.pone.0062957- Yang J, Zhang C-T, Yuan X-J, et al. Metabolic engineering of
     Methylobacterium extorquens AM1 for the production of butadiene precursor.
     *Microb Cell Fact*. 2018;17(1):194. doi:10.1186/s12934-018-1042-4- Singh HB, Kang M-K, Kwon M, Kim S-W. Developing methylotrophic microbial
       platforms for a methanol-based bioindustry. *Front Bioeng Biotechnol*.
       2022;10. doi:10.3389/fbioe.2022.1050740- Schada von Borzyskowski L, Remus-Emsermann M, Weishaupt R, Vorholt JA, Erb
         TJ. A set of versatile brick vectors and promoters for the assembly, expression,
         and integration of synthetic operons in Methylobacterium extorquens AM1 and
         other alphaproteobacteria. *ACS Synth Biol*. 2015;4(4):430-443.
         doi:10.1021/sb500221v- Carrillo M, Wagner M, Petit F, Dransfeld A, Becker A, Erb TJ. Design and
           Control of Extrachromosomal Elements in Methylorubrum extorquens AM1. *ACS
           Synth Biol*. 2019;8(11):2451-2456. doi:10.1021/acssynbio.9b00220- Kaczmarczyk A, Vorholt JA, Francez-Charlot A. Synthetic vanillate-regulated
             promoter for graded gene expression in Sphingomonas. *Sci Rep*.
             2014;4:6453. doi:10.1038/srep06453- Leveau JHJ, Lindow SE. Predictive and Interpretive Simulation of Green
               Fluorescent Protein Expression in Reporter Bacteria. *J Bacteriol*.
               2001;183(23):6752-6762. doi:10.1128/JB.183.23.6752-6762.2001- Orita I, Nishikawa K, Nakamura S, Fukui T. Biosynthesis of
                 polyhydroxyalkanoate copolymers from methanol by Methylobacterium extorquens AM1
                 and the engineered strains under cobalt-deficient conditions. *Appl Microbiol
                 Biotechnol*. 2014;98(8):3715-3725. doi:10.1007/s00253-013-5490-9- Thomson NM, Saika A, Ushimaru K, et al. Efficient production of active
                   polyhydroxyalkanoate synthase in Escherichia coli by coexpression of molecular
                   chaperones. *Appl Environ Microbiol*. 2013;79(6):1948-1955.
                   doi:10.1128/AEM.02881-12- https://nebuilder.neb.com/#!/.- Nile red: a selective fluorescent stain for intracellular lipid droplets.
                       *J Cell Biol*. 1985;100(3):965-973.- Zuriani R, Vigneswari S, Azizan MNM, Majid MIA, Amirul AA. A high throughput
                         Nile red fluorescence method for rapid quantification of intracellular bacterial
                         polyhydroxyalkanoates. *Biotechnol Bioproc E*. 2013;18(3):472-478.
                         doi:10.1007/s12257-012-0607-z- Suzuki T, Yamane T, Shimizu S. Kinetics and effect of nitrogen source
                           feeding on production of poly-?-hydroxybutyric acid by fed-batch culture.
                           *Appl Microbiol Biotechnol*. 1986;24(5):366-369. doi:10.1007/BF00294591- Bourque D, Pomerleau Y, Groleau D. High-cell-density production of
                             poly-β-hydroxybutyrate (PHB) from methanol by Methylobacterium extorquens:
                             production of high-molecular-mass PHB. *Appl Microbiol Biotechnol*.
                             1995;44(3-4):367-376. doi:10.1007/BF00169931- Cardoso LOB, Karolski B, Gracioso LH, do Nascimento CAO, Perpetuo EA.
                               Increased P3HB Accumulation Capacity of Methylorubrum sp. in Response to
                               Discontinuous Methanol Addition. *Appl Biochem Biotechnol*.
                               2020;192(3):846-860. doi:10.1007/s12010-020-03369-9- Sonicated PHA beads.
                                 https://www.sigmaaldrich.com/NL/en/product/aldrich/363502?gclid=CjwKCAjw4P6oBhBsEiwAKYVkq9Sz8n\_xmsFZOZUTSgJyGOATxkkHN8kl8-\_Gx0iRCudNGYpkvbgV4BoCBT4QAvD\_BwE- van Dien SJ, Marx CJ, O'Brien BN, Lidstrom ME. Genetic Characterization of
                                   the Carotenoid Biosynthetic Pathway in Methylobacterium extorquens AM1 and
                                   Isolation of a Colorless Mutant. *Appl Environ Microbiol*.
                                   2003;69(12):7563-7566. doi:10.1128/AEM.69.12.7563-7566.2003- Mo X-H, Zhang H, Wang T-M, et al. Establishment of CRISPR interference in
                                     Methylorubrum extorquens and application of rapidly mining a new phytoene
                                     desaturase involved in carotenoid biosynthesis. *Appl Microbiol
                                     Biotechnol*. 2020;104(10):4515-4532. doi:10.1007/s00253-020-10543-w- De Novo DNA. https://www.denovodna.com/software/- Zha J, Liu Z, Sun R, Gong G, Dordick JS, Wu X. Endolysin-Based Autolytic E.
                                         coli System for Facile Recovery of Recombinant Proteins. *J Agric Food
                                         Chem*. 2021;69(10):3134-3143. doi:10.1021/acs.jafc.1c00059- Borrero-de Acuña JM, Hidalgo-Dumont C, Pacheco N, Cabrera A, Poblete-Castro
                                           I. A novel programmable lysozyme-based lysis system in Pseudomonas putida for
                                           biopolymer production. *Sci Rep*. 2017;7(1):4373.
                                           doi:10.1038/s41598-017-04741-2- Attai H, Rimbey J, Smith GP, Brown PJB. Expression of a Peptidoglycan
                                             Hydrolase from Lytic Bacteriophages Atu\_ph02 and Atu\_ph03 Triggers Lysis of
                                             Agrobacterium tumefaciens. *Appl Environ Microbiol*. 2017;83(23).
                                             doi:10.1128/AEM.01498-17- Rodríguez-Rubio L, Gerstmans H, Thorpe S, Mesnage S, Lavigne R, Briers Y.
                                               DUF3380 Domain from a Salmonella Phage Endolysin Shows Potent N-Acetylmuramidase
                                               Activity. *Appl Environ Microbiol*. 2016;82(16):4975-4981.
                                               doi:10.1128/AEM.00446-16- Haro A, Vélez M, Goormaghtigh E, et al. Reconstitution of holin activity
                                                 with a synthetic peptide containing the 1-32 sequence region of EJh, the EJ-1
                                                 phage holin. *J Biol Chem*. 2003;278(6):3929-3936.
                                                 doi:10.1074/JBC.M211334200- Hu B, Yang Y-M, Beck DAC, et al. Comprehensive molecular characterization of
                                                   Methylobacterium extorquens AM1 adapted for 1-butanol tolerance. *Biotechnol
                                                   Biofuels*. 2016;9:84. doi:10.1186/S13068-016-0497-Y- Koller M, Niebelschütz H, Braunegg G. Strategies for recovery and
                                                     purification of poly[(R )‐3‐hydroxyalkanoates] (PHA) biopolyesters from
                                                     surrounding biomass. *Engineering in Life Sciences*. 2013;13(6):549-562.
                                                     doi:10.1002/elsc.201300021- Mongili B, Abdel Azim A, Fraterrigo Garofalo S, et al. Novel insights in
                                                       dimethyl carbonate-based extraction of polyhydroxybutyrate (PHB). *Biotechnol
                                                       Biofuels*. 2021;14(1):13. doi:10.1186/s13068-020-01849-y

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