iGEM 2022 — Team ionis-paris

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{% block title %}Notebook{% endblock %}
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# Notebook

We organize our notebook per week. Let’s see how we conducted our laboratory work.

### First week

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* [Week 1](https://static.igem.wiki/teams/4187/wiki/notebook/semaine-1.pdf)

### Second week

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* [Week 2](https://static.igem.wiki/teams/4187/wiki/notebook/semaine-2.pdf)

### Third week

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* [Week 3](https://static.igem.wiki/teams/4187/wiki/notebook/semaine-3.pdf)

### Fourth week

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* [Week 4](https://static.igem.wiki/teams/4187/wiki/notebook/semaine-4.pdf)

### Fifth week

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* [Week 5](https://static.igem.wiki/teams/4187/wiki/notebook/semaine-5.pdf)

### Sixth week

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* [Week 6](https://static.igem.wiki/teams/4187/wiki/notebook/semaine-6.pdf)

### Seventh week

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* [Week 7](https://static.igem.wiki/teams/4187/wiki/notebook/semaine-7.pdf)

### Eight week

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* [Week 8](https://static.igem.wiki/teams/4187/wiki/notebook/semaine-8.pdf)

### Ninth week

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* [Week 9](https://static.igem.wiki/teams/4187/wiki/notebook/semaine-9.pdf)

### Tenth week

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* [Week 10](https://static.igem.wiki/teams/4187/wiki/notebook/semaine-10.pdf)

### Eleventh week

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* [Week 11](https://static.igem.wiki/teams/4187/wiki/notebook/semaine-11.pdf)

### Twelfth week

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* [Week 12](https://static.igem.wiki/teams/4187/wiki/notebook/semaine-12.pdf)

### Thirteenth week

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* [Week 13](https://static.igem.wiki/teams/4187/wiki/notebook/semaine-13.pdf)

### Fourteenth week

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* [Week 14](https://static.igem.wiki/teams/4187/wiki/notebook/semaine-14.pdf)

### Fifteenth week

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* [Week 15](https://static.igem.wiki/teams/4187/wiki/notebook/semaine-15.pdf)

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# Engineering Success

For the Engineering Success part, we focused on the alpha-amylase (AmyH) part: BBa\_K4187022
composed of the AmyH coding sequence BBa\_K4187016, the T7 promoter BBa\_I719005, the
strong RBS.1 BBa\_B0030 and the double terminator BBa\_B0015.

## Cycle 1

### Design:

First, we designed our sequence of the alpha-amylase used (AmyH) from the bacterium
*Halomonas meridiana* on the SnapGene software (see Figure 1). We added the PstI and SacI
restriction sites to the coding sequence ends to be able to extract and clone the sequence and
further assembly. We want to overexpress an amylase in *Escherichia coli* to degrade the remaining
starch in the brewers’ spent grain and to produce glucose.

![Sequence of the initial composite part of AmyH with the T7 promoter](https://static.igem.wiki/teams/4187/wiki/engineering-success/engineering-img-1.jpg)

**Figure 1: Sequence of the initial composite part of AmyH with the T7 promoter, the strong Ribosome Binding Site (RBS) and the double terminator made in SnapGene.**
The size of the sequence was 1634bp.

### Build:

Once we received our sequence in a pUC19 plasmid from Integrated DNA Technologies
(IDT), we transformed *E. Coli DH5α* to amplify it and bank it.

### Test:

Then, to check our sequence of interest, we did a mini-prep, followed by a digestion using
the restriction enzymes SacI and PstI, and ended by an electrophoresis of the digestion. As
expected, we obtained two bands (see Figure 2). Once checked, we transformed *E. Coli BL21* to
try a functional characterisation by performing a Lugol test on Petri dishes with a non-transformed
*E. coli BL21* negative control and with a transformed *E. coli* BL21 with AmyH.

![The AmyH digested with SacI and PstI](https://static.igem.wiki/teams/4187/wiki/engineering-success/engineering-img-2.png)

**Figure 2: The AmyH digested with SacI and PstI.**
The DNA ladder used is the 1 kb DNA Ladder from Promega that is defined on the left. The
expected size of the AmyH was 1,614 bp. The observed bands are above 1,500bp. These bands
correspond to the expected size of the corrected AmyH.

### Learn:

During our functional characterisation, we found no significant effect from AmyH compared
to our control. Thus, we decided to check our designed sequence. We found that there was an
issue with our T7 promoter: it was truncated (see Figure 3). The amylase could not be expressed.
In fact, our truncated T7 promoter size was 10bp instead of 23bp.

![Comparison of our truncated T7 promoter with the T7 promoter](https://static.igem.wiki/teams/4187/wiki/engineering-success/engineering-img-3.png)

**Figure 3: Comparison of our truncated T7 promoter with the T7 promoter made on Benchling**

## Cycle 2

### Design:

To fix our promoter, we designed and ordered primers to be able to fix the issue on the
alpha-amylase with the truncated T7 promoter via Polymerase Chain Reaction (PCR) (see Figure 4).

![Sequences of our Forward and Reverse primers to correct T7 promoter of AmyH](https://static.igem.wiki/teams/4187/wiki/engineering-success/engineering-img-4.png)

**Figure 4: Sequences of our Forward and Reverse primers to correct T7 promoter of AmyH done on SnapGene**

![The fixation of both primers, forward and reverse, on the AmyH composite part with the truncated T7 promoter 1](https://static.igem.wiki/teams/4187/wiki/engineering-success/engineering-img-5-1.png)
![The fixation of both primers, forward and reverse, on the AmyH composite part with the truncated T7 promoter 2](https://static.igem.wiki/teams/4187/wiki/engineering-success/engineering-img-5-2.png)

![The fixation of both primers, forward and reverse, on the AmyH composite part with the truncated T7 promoter 3](https://static.igem.wiki/teams/4187/wiki/engineering-success/engineering-img-5-3.png)

**Figure 5: The fixation of both primers, forward and reverse, on the AmyH composite part with the truncated T7 promoter from SnapGene.**
We deleted the parts that we had originally planned for the Golden Gate assembly, so we expect to see a band at 1629pb.

### Build:

We performed a PCR on the alpha-amylase with the truncated T7 promoter, and analyzed
it by electrophoresis. We used the protocols: Polymerase Chain Reaction using Q5® High-Fidelity
2X Master Mix of NEB and Electrophoresis (Gel preparation and migration) in the
[Experiments part.](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)

### Test:

The electrophoresis gel showed the expected bands of AmyH, *1,629 bp*, (see Figure 6), so
we tried once again to assemble it in the pmScarlet-i\_C1 (Plasmid #85044 on Addgene)
[2] plasmid (see Figure 7 and 8) to try our functional characterisation once again.

![Gel electrophoresis of the corrected amplified AmyH and LDH](https://static.igem.wiki/teams/4187/wiki/engineering-success/engineering-img-6.png)

**Figure 6: Gel electrophoresis of the corrected amplified AmyH and LDH done on 09/27/2022.**
The DNA ladder used is the 1 kb Plus DNA Ladder from NEB that is defined on the left. Both T7
promoters of LDH and AmyH were corrected via PCR. Without counting the last well, the first six
wells correspond to AmyH. The expected size of the AmyH was 1,629 bp. The observed bands
are slightly above 1,500bp. These bands correspond to the expected size of the corrected AmyH.
In the fifth well of AmyH, the PCR did not work probably because of the absence of DNA.

![pmScarlet-i_C1 plasmid used as a vector for the assembly of AmyH](https://static.igem.wiki/teams/4187/wiki/engineering-success/engineering-img-7.png)

**Figure 7: pmScarlet-i\_C1 plasmid used as a vector for the assembly of AmyH.**
This backbone has the two restriction sites SacI and PstI to assemble correctly AmyH digested
with those restriction enzymes

![Picture of the Petri dish with the ligation of digested AmyH and digested](https://static.igem.wiki/teams/4187/wiki/engineering-success/engineering-img-8.png)

**Figure 8: Picture of the Petri dish with the ligation of digested AmyH and digested pmScarlet-i\_C1 plasmid.**
The colonies are pink because the Red Fluorescent Protein (RFP) is still expressed in our ligation.

### Learn:

This time, the characterisation yielded good results (see Figure 9) (see Proof of concept for
more). Therefore, we attempted to assemble our whole Depolymerization plasmid (see Figure 7,
Parts collection).

![Pictures of our characterization of AmyH](https://static.igem.wiki/teams/4187/wiki/engineering-success/engineering-img-9.jpg)

**Figure 9: Pictures of our characterization of AmyH under the same conditions with E.Coli BL21 transformed with pmScarlet-i\_C1 plasmid containing the AmyH composite part and untransformed E.Coli BL21 (wt).**
Salt was added because the amylase that we chose has an
optimal activity with salt [1]. We dyed the starch with a lugol solution. Starch concentration: 0.2 g/L
NaCl concentration: 5% (w/v) NT BL21 mean halo surface = 0.05 cm²; amyH BL21 mean halo
surface = 1.25 cm²

## Cycle 3

### Design:

We had decided to assemble the Depolymerization plasmid in the pSB1A3 plasmid, and
had chosen the appropriate enzymes to perform the Digestion-Ligation assembly.

### Build:

In our Depolymerization plasmid, we had three inserts and one plasmid to digest. AmyH
was once again digested with SacI and Pst1.

### Test:

We tested our digestion by electrophoresis to check our ligation. We obtained the
appropriate bands on our electrophoresis, but when we purified our gel, we obtained weak
concentration (>5 ng/uL). We still tried to perform our ligation, but it wasn't successful as nothing
grew on the Petri Dish (see Figure 10).

![Picture of a Petri dish containing E.Coli BL21 with the Depolymerisation plasmid](https://static.igem.wiki/teams/4187/wiki/engineering-success/engineering-img-10.png)

**Figure 10: Picture of a Petri dish containing E.Coli BL21 with the Depolymerisation plasmid.**
No colony is observed. The ligation did not work.

### Learn:

We learned that we have to try again the ligation by increasing the concentration of the
vector and the inserts. To do that, we will try other ratios as 1 vector:1 insert, 1 vector: 2 inserts for
the ligation. We will do other mini preps of pSB1A3, EL222, PLAase, AmyH to have a higher
quantity of plasmid and inserts. If it does not work, we should order primers to amplify our purified
fragments,the plasmid and the inserts, to have a higher concentration.

These cycles permit the correction of a few issues. However, our last engineering success
cycle was not successful, other cycles will probably be needed to succeed the depolymerization
plasmid assembly.

## References

1. María-José Coronado, Carmen Vargas, Jürgen Hofemeister, Antonio Ventosa, Joaquín J.
   Nieto, Production and biochemical characterization of an α-amylase from the moderate
   halophile *Halomonas meridiana*, *FEMS Microbiology Letters*, Volume 183, Issue 1, February
   2000, Pages 67–71.
2. mScarlet: a bright monomeric red fluorescent protein for cellular imaging. Bindels DS,
   Haarbosch L, van Weeren L, Postma M, Wiese KE, Mastop M, Aumonier S, Gotthard G, Royant A,
   Hink MA, Gadella TW Jr. *Nat Methods*. 2016 Nov 21

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# Experiments

Please find out below the list of the protocols that we conducted:

### Potency of DNA using QubitTM from ThermoFisher

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* [Potency of DNA using QubitTM from ThermoFisher](https://static.igem.wiki/teams/4187/wiki/experiments/potency-of-dna-using-a-qubit-from-thermofisher.pdf)

### Electrophoresis (Gel preparation and migration)

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* [Electrophoresis (Gel preparation and migration)](https://static.igem.wiki/teams/4187/wiki/experiments/electrophoresis-gel-preparation-and-migration.pdf)

### Precipitation of DNA with isopropanol

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* [Precipitation of DNA with isopropanol](https://static.igem.wiki/teams/4187/wiki/experiments/precipitation-of-dna-with-isopropanol.pdf)

### DNA miniprep

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* [DNA miniprep using Monarch® kit from NEB](https://static.igem.wiki/teams/4187/wiki/experiments/dna-miniprep-using-monarch-kit-from-neb.pdf)
* [DNA miniprep using Wizard® Plus SV Minipreps DNA Purification System from Promega](https://static.igem.wiki/teams/4187/wiki/experiments/dna-miniprep-using-wizard-plus-sv-minipreps-dna-purification-system-from-promega.pdf)

### Digestion with restriction enzymes

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* [Digestion with restriction enzymes](https://static.igem.wiki/teams/4187/wiki/experiments/digestion-with-restriction-enzymes.pdf)

### Gel Extraction by using QIAquick Kit from Qiagen

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* [Gel Extraction by using QIAquick Kit from Qiagen](https://static.igem.wiki/teams/4187/wiki/experiments/gel-extraction-by-using-qiaquick-kit-from-qiagen.pdf)

### Ligation protocol using NEB reagents

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* [Ligation protocol using NEB reagents](https://static.igem.wiki/teams/4187/wiki/experiments/ligation-protocol-using-neb-reagents.pdf)

### Competent bacteria

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* [Competent bacteria](https://static.igem.wiki/teams/4187/wiki/experiments/competent-bacteria.pdf)

### Transformation of competent bacteria

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* [Transformation of competent bacteria](https://static.igem.wiki/teams/4187/wiki/experiments/competent-bacteria-transformation.pdf)

### Polymerase Chain Reaction

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* [Polymerase Chain Reaction using GoTaq® Long PCR Master Mix of Promega](https://static.igem.wiki/teams/4187/wiki/experiments/polymerase-chain-reaction-using-gotaq-long-pcr-master-mix-of-promega.pdf)
* [Polymerase Chain Reaction using Q5® High-Fidelity 2X Master Mix of NEB](https://static.igem.wiki/teams/4187/wiki/experiments/polymerase-chain-reaction-using-q5-high-fidelity-2x-master-mix-of-neb.pdf)

### Preparation of miniculture

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* [Preparation of miniculture](https://static.igem.wiki/teams/4187/wiki/experiments/preparation-of-mini-culture.pdf)

### Preparation of Petri dishes

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* [Preparation of Petri dishes](https://static.igem.wiki/teams/4187/wiki/experiments/preparation-of-petri-dishes.pdf)

### Colony PCR

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* [Colony PCR](https://static.igem.wiki/teams/4187/wiki/experiments/colony-pcr.pdf)

### Plate Nile red staining

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* [Plate Nile red staining](https://static.igem.wiki/teams/4187/wiki/experiments/plate-nile-red-staining.pdf)

### Assessment of the production of D-Lactate by using D-Lactate Assay Kit from Sigma Aldrich

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* [Assessment of the production of D-Lactate by using D-Lactate Assay Kit from Sigma Aldrich](https://static.igem.wiki/teams/4187/wiki/experiments/assessment-of-the-production-of-d-lactate-by-using-d-lactate-assay-kit-from-sigma-aldrich.pdf)

### Preparation of M9 minimal media for *Escherichia coli*

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* [Preparation of M9 minimal media for *Escherichia coli*](https://static.igem.wiki/teams/4187/wiki/experiments/preparation-of-m9-minimal-media-for-escherichia-coli.pdf)

### Electrophoresis of proteins (SDS-Page from bacterial cells)

---

* [Electrophoresis of proteins (SDS-Page from bacterial cells)](https://static.igem.wiki/teams/4187/wiki/experiments/electrophoresis-of-proteins-sds-page-from-bacterial-cells.pdf)

## References

1. Elbing KL, Brent R, 2019. Recipes and Tools for Culture of Escherichia coli. *Curr Protoc Mol
   Biol.* 25(1)
2. Merk, 2022. *D-Lactate Assay Kit*. Available at:
   <https://www.sigmaaldrich.com/FR/fr/product/sigma/mak336> (accessed on 10/06/2022)
3. New England Biolabs, 2022. *Ligation Protocol with T4 DNA Ligase (M0202)*. Available at:
   <https://international.neb.com/protocols/0001/01/01/dna-ligation-with-t4-dna-ligase-m0202>
   (accessed on 09/24/2022)
4. New England Biolabs, 2022. *Monarch® Kits for Plasmid Purification*. Available at:
   <https://international.neb.com/monarch/monarch-kits-for-plasmid-purification> (accessed on
   09/24/2022)
5. New England Biolabs, 2022. *Protocol for Q5® High-Fidelity 2X Master Mix*. Available at:
   <https://international.neb.com/protocols/2012/12/07/protocol-for-q5-high-fidelity-2x-master-mix-m0492>
   (accessed on 09/24/2022)
6. Promega, 2022. *Long-Range PCR: GoTaq® Long PCR Master Mix*. Available at:
   <https://france.promega.com/products/pcr/taq-polymerase/gotaq-long-range-pcr-master-mix/?catNum=M4021#protocols>
   (accessed on 09/24/2022)
7. Promega, 2022. *Wizard® Plus SV Minipreps DNA Purification Systems*. Available at:
   <https://france.promega.com/en/products/nucleic-acid-extraction/plasmid-purification/wizard-plus-sv-minipreps-dna-purification-systems/?catNum=A1330>
   (accessed on 09/24/2022)

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# Results

## 1. Construction of our plasmids of interest

At first sight, we decided to do the Golden Gate assembly but we decided to choose
a simple method. For the assembly of our BioBricks, we did a method that is widely used:
the BioBrick Assembly (Restriction Enzymes) method. For that, we designed our different
coding sequences with the basic parts (Ribosome Binding Site (RBS), a T7 promoter, a
double terminator) and with the restriction enzyme sites we wanted.

### a. Polymerization Plasmid

For the polymerization plasmid ([http://parts.igem.org/Part:BBa\_K4187029](http://parts.igem.org/Part%3ABBa_K4187029)),
corresponding to pSB1K3) , our BioBrick is composed of the following composite parts: a
D-Lactate Dehydrogenase (LDH) ([http://parts.igem.org/Part:BBa\_K4187017](http://parts.igem.org/Part%3ABBa_K4187017)), followed by a
propionate-CoA transferase (Pct) ([http://parts.igem.org/Part:BBa\_K4187018](http://parts.igem.org/Part%3ABBa_K4187018)) and a
Polyhydroxyalkanoate (PHA) synthase (PHAC1) ([http://parts.igem.org/Part:BBa\_K4187019](http://parts.igem.org/Part%3ABBa_K4187019)).

Our first step was the digestion of each of our gene fragments with the corresponding
restriction enzymes. LDH was digested with XbaI and BamHI. Pct was digested with BamHI
and SacI. PHAC1 was digested with SacI and PstI. The pSB1K3 was digested with Xba1
and Pst1 (see Figure 1).

![Gel electrophoresis of a migration of PHAC1, PCT, pSB1K3](https://static.igem.wiki/teams/4187/wiki/results/results-img-1.png)

**Figure 1: Gel electrophoresis of a migration of PHAC1, PCT, pSB1K3 done on 09/05/2022.**
The DNA ladder is a 1kb DNA Ladder from Promega defined on the left. The
first three wells correspond to PHAC1 digested with SacI and PstI. The next two wells
correspond to Pct digested with BamHI and SacI. The next three wells correspond to
pSB1K3 digested with XbaI and PstI. For both PHAC1 and Pct, the expected size was
1,896bp. The observed bands are below 2,000bp corresponding to the expected size of our
inserts. pSB1K3 was also correctly digested.

For LDH, we encountered two problems in our sequences: there was a methylation
on XbaI restriction site and the T7 promoter did not have all of its sequence. To correct it, we ordered
primers and we did a Polymerase Chain Polymerase (PCR).

![Gel electrophoresis of the corrected amplified LDH](https://static.igem.wiki/teams/4187/wiki/results/results-img-2.png)

**Figure 2: Gel electrophoresis of the corrected amplified LDH done on 09/27/2022.**
The DNA ladder used is the 1 kb Plus DNA Ladder from NEB that is defined on
the left. LDH was corrected on both issues: the methylation and the T7 promoter. Without
counting the last well, the last three wells correspond to the amplified LDH. The expected
size of the LDH was 1,243 bp. The observed bands are slightly above 1,200bp. These
bands correspond to the expected size of the corrected LDH.

Once corrected, we have to digest the amplified fragment of LDH with the restriction
enzymes: XbaI and BamHI. However, we did not have any DNA ladder that could permit us
to check the digestion of LDH in our stock.

![Expected result from the amplified LDH digested from Benching](https://static.igem.wiki/teams/4187/wiki/results/results-img-3.png)

LDH is digested by XbaI and BamHI in silico. The expected size is 1,224bp. The other bands are at
12bp and 7bp.

![Gel electrophoresis of the digested LDH obtained from the amplified LDH](https://static.igem.wiki/teams/4187/wiki/results/results-img-4.png)

**Figure 4: Gel electrophoresis of the digested LDH obtained from the amplified LDH done on 09/30/22.**
LDH is digested by XbaI and BamHI. The DNA ladder used is the 1 kb
Plus DNA Ladder from NEB. The expected size is 1,224bp. The other bands are at 12bp
and 7bp. The other two bands are not observable on this gel.

Because of the impossibility of checking the digestion of the corrected LDH, we
directly tried the assembly of our polymerization BioBrick.

### b. Depolymerization plasmid

For the depolymerization plasmid ([http://parts.igem.org/Part:BBa\_K4187024](http://parts.igem.org/Part%3ABBa_K4187024))
corresponding to pSB1A3, our BioBrick is composed of Light-activated DNA-binding protein
(EL222) ([http://parts.igem.org/Part:BBa\_K4187020](http://parts.igem.org/Part%3ABBa_K4187020)), followed by the Blue light inducible
promoter (Pblind), an inducible promoter, that is activated by EL222 inducing the expression
of a Polylactate depolymerase (PLAase) ([http://parts.igem.org/Part:BBa\_K4187021](http://parts.igem.org/Part%3ABBa_K4187021)), and
then completed with an alpha-amylase (AmyH) ([http://parts.igem.org/Part:BBa\_K4187022](http://parts.igem.org/Part%3ABBa_K4187022)).

The purpose was also to make this construction by also using the restriction enzyme
method. EL222 was digested with XbaI and NcoI. PLAase was digested with NcoI and SacI.
AmyH was digested with SacI and PstI.

![Gel electrophoresis of a migration of EL222, pSB1A3](https://static.igem.wiki/teams/4187/wiki/results/results-img-5.png)

**Figure 5: Gel electrophoresis of a migration of EL222, pSB1A3 done on 08/26/2022.**
The DNA ladder is a 1kb DNA Ladder from Promega. The first three wells
correspond to EL222 digested with XbaI and NcoI. The next two wells correspond to
pSB1A3 digested with XbaI and PstI. EL222 was not digested. pSB1A3 was correctly
digested mainly on the third well because three bands were observed. Xba1 cut at two sites.
The correct band is the above one at 2,053bp.

![Expected result from the pSB1A3 digested from Benching](https://static.igem.wiki/teams/4187/wiki/results/results-img-6.png)

**Figure 6: Expected result from the pSB1A3 digested from Benching.**
pSB1A3 is digested by XbaI and PstI in silico. The expected size is 2,053bp. The other bands are at
1,329bp and 948bp.

For these results on EL222, we checked the sequence and we saw a methylation on
the XbaI site preventing the cut by XbaI. We ordered the correct sequence without the
methylation. However, we received EL222 as a gene fragment. So we could not check if it
was correctly digested or not.

![Gel electrophoresis of a migration of the correct EL222](https://static.igem.wiki/teams/4187/wiki/results/results-img-7.png)

**Figure 7: Gel electrophoresis of a migration of the correct EL222 digested on 09/21/22.**
The DNA ladder used is the 1 kb Plus DNA Ladder from NEB.Without counting the
DNA ladder, the third well corresponds to EL222 digested with XbaI and NcoI. Digested
EL222 length is 929bp. The other bands are not observable but they are at 8bp and 12bp.

![Expected result from the EL222 digested from Benching](https://static.igem.wiki/teams/4187/wiki/results/results-img-8.png)

**Figure 8: Expected result from the EL222 digested from Benching.**
EL222 is digested by XbaI and NcoI in silico. The expected size EL222 length is 929bp. The other
bands are at 8bp and 12bp.

As LDH, we could not check the digestion, so we tried directly with the purified band
of EL222 obtained for the assembly.

![Gel electrophoresis of a migration of the digested PLAase](https://static.igem.wiki/teams/4187/wiki/results/results-img-9.png)

**Figure 9: Gel electrophoresis of a migration of the digested PLAase done on 09/21/22.**
The DNA ladder used is the 1 kb Plus DNA Ladder from NEB. Without counting the
DNA ladder, the second well corresponds to PLAase digested with NcoI and SacI. Digested
PLAase length is 1,179bp.

For AmyH, we encountered the same problem with the T7 promoter, so we had to
correct it via PCR. We designed the primers for it.

![Gel electrophoresis of the corrected amplified AmyH](https://static.igem.wiki/teams/4187/wiki/results/results-img-10.png)

**Figure 10: Gel electrophoresis of the corrected amplified AmyH done on 09/27/2022.**
The DNA ladder used is the 1 kb Plus DNA Ladder from NEB. The T7 promoter
of AmyH was corrected. Without counting the DNA ladder, the first six wells correspond to
the amplified AmyH. The expected size of the AmyH was 1,629 bp. The observed bands are
slightly above 1,500bp. These bands correspond to the expected size of the corrected
AmyH.

Then, we digested the amplified AmyH. But, we encountered the same problem as
LDH. We could not check that our amplified AmyH is digested.

![Expected result from the amplified AmyH digested from Benching](https://static.igem.wiki/teams/4187/wiki/results/results-img-11.png)

**Figure 11: Expected result from the amplified AmyH digested from Benching.**
AmyH is digested by SacI and PstI. The expected size is 1,610bp. The other bands are at 8bp and
11bp.

![Gel electrophoresis of the digested AmyH obtained from the amplified AmyH](https://static.igem.wiki/teams/4187/wiki/results/results-img-12.png)

**Figure 12: Gel electrophoresis of the digested AmyH obtained from the amplified AmyH done on 09/30/22.**
The DNA ladder used is the 1 kb Plus DNA Ladder from NEB.
AmyH is digested by SacI and PstI. The expected size is 1,610bp. The other bands are at
12bp and 7bp. The other two bands are not observable on this gel.

Once AmyH is digested, we tried the assembly of the depolymerization plasmid as well as
the polymerization plasmid.

However, we did not succeed to do our assemblies.

## 2. *Alpha-amylase* (AmyH) functional assay

We assessed the ability of *E.Coli* BL21 transformed with the amyH (amyH BL21)
gene to degrade starch by performing a lugol staining assay on solid cultures. As this
amylase’s activity depends on NaCl concentrations, we tested multiple salt concentrations
(i.e. 0%, 5%). We cultured the bacteria as well-defined separated colonies on solid media
containing starch. We defined degradation halos as the total unstained surface minus the
surface of the colony: S(unstained) - S(colony).

We measured these surfaces using ImageJ, an open source software developed by Wayne
Rasband.

![Picture of Lugol staining assay comparing wt BL21 to amyH BL21](https://static.igem.wiki/teams/4187/wiki/results/results-img-13.jpg)

**Figure 13: Picture of Lugol staining assay comparing wt BL21 to amyH BL21.**
Starch concentration: 0.2 g/L NaCl concentration: 0% (w/v) NT BL21 mean halo surface = 0.7 cm²;
amyH BL21 mean halo surface = 1.28 cm²

![Picture of Lugol staining assay comparing wt BL21 to amyH BL21](https://static.igem.wiki/teams/4187/wiki/results/results-img-14.jpg)

**Figure 14: Picture of Lugol staining assay comparing wt BL21 to amyH BL21.**
Starch concentration: 0.2 g/L NaCl concentration: 5% (w/v) NT BL21 mean halo surface = 0.05 cm²;
AmyH BL21 mean halo surface = 1.25 cm²

Our results show an increased starch degradation activity in *E.coli* BL21 transformed with
AmyH as the mean halo sizes of the colonies are higher in transformed colonies.
We cannot conclude on the NaCl concentration effect on the amylase activity as the
maximum mean halo sizes are similar in both NaCl concentration conditions. However we
observe that with a 5% NaCl concentration, halo sizes of non-transformed *BL21* are close to
zero while halo sizes of transformed *BL21* reach the same value as, in the 0% NaCl
condition.

## 3. *Shewanella oneidensis* electric activity

We tested the ability of *Shewanella oneidensis* to generate an electric current
in varying conditions. We tested the effect of lactate, riboflavin, and the mtrC gene
overexpression against a non-transformed *Shewanella oneidensis* without any lactate in the
medium. We performed these measurements using our first prototype described in Figure
15. As our hardware was not sensitive enough to measure intensity in our working ranges,
we decided to measure voltage and made the assumption that our system’s resistance
remained the same throughout the experiment.

![Prototype used for the measurements](https://static.igem.wiki/teams/4187/wiki/results/results-img-15.png)

**Figure 15: Prototype used for the measurements.**
Two 50mL tubes filled with 30mL LB
growth medium are separated by a proton exchange membrane. Electrodes are made of
carbon fiber. Iron electric wires connect the electrodes to a multimeter. The composition of
the first chamber varies depending on the tested conditions.

![Records of the tension variation across the system over a period of 9 hours with varying conditions](https://static.igem.wiki/teams/4187/wiki/results/results-img-16.png)

**Figure 16: Records of the tension variation across the system over a period of 9 hours with varying conditions.**
In light blue: wild type Shewanella oneidensis; in yellow:
wild type shewanella oneidensis supplemented with 10mM lactate; in grey Shewanella
Oneidensis transformed with mtrC and supplemented with 10mM lactate; in orange:
Shewanella Oneidensis transformed with mtrC and supplemented with 10mM lactate and
37nM riboflavin; in deep blue: Co-culture of Shewanella oneidensis transformed with mtrC
and E.coli BL21 transformed with phenazine-1-carboxylic acid coding gene and
supplemented with 10mM lactate.

All conditions plateaued at their maximum tension variation 20 to 30 minutes after the
beginning of the experience. The maximum tension variation of 28mV was observed with
*Shewanella oneidensis* transformed with the mtrC gene and supplemented with 37nM
riboflavin. This shows us that riboflavin might be the best way for improving electron transfer
as it reaches a tension of over 10 mV above all the other conditions.

No significant difference was observed between *Shewanella oneidensis* transformed with
mtrC and non-transformed *Shewanella oneidensis* which both plateau at around 16mV.
*Shewanella oneidensis* transformed with p150 plateaued at around 5mV. Non
lactate-supplemented wild type shewanella oneidensis did not show any consistent tension
plateau and showed a max tension of 3mV. After 9 hours, all MFC returned to their baseline
tension.

The results from the co-culture of *E.coli* secreting Phenazine-1-carboxylic acid and
*Shewanella oneidensis* were not what we were expecting, especially compared to
*Shewanella oneidensis* with Riboflavin. Indeed, we expected to see better results coming
from the Phenazine-1-carboxylic acid. However, the test with Phenazine-1-carboxylic acid
involved a co-culture of *Shewanella oneidensis* and *E. coli*, and we suspect that this is why
we obtained weak results. We believe that *E. coli* may have overgrew
*Shewanella oneidensis*.

## 4. Checking the expression of our proteins

First, we performed a cell lysis to extract the proteins. Then, to obtain the
concentrations of our samples, we carried out a standard curve by using Bradford reagent
measuring the absorbance of known concentrations of BSA. (see Figure 17)

![Standard curve from BSA proteins from 0 to 2g/L](https://static.igem.wiki/teams/4187/wiki/results/results-img-17.png)

**Figure 17: Our standard curve from BSA proteins from 0 to 2g/L to calculate the concentration of proteins by using Bradford assay by measuring the absorbance.**
Created by using different solutions with various concentrations of BSA and measured at
595nm by spectrophotometer.

Once we got this curve, we were able to get the concentration of our samples by measuring
their absorbance. Depending on their concentration, the samples were diluted by 2 or by 5.
Then, in order to get optimal results in our SDS-Page, we wanted 20 ug of proteins per well.
The results can be seen in Figure 18.

![Table showing the concentration of our different samples and the corresponding volume for the SDS-Page](https://static.igem.wiki/teams/4187/wiki/results/results-img-18.png)

**Figure 18: Table showing the concentration of our different samples and the corresponding volume for the SDS-Page.**
Each sample's absorbance was measured at
595 nm and the concentration was calculated by taking into account the dilution factor. Then,
we calculated the volume needed to get 20ug of proteins in each of our wells.

After checking the amount of proteins in our samples, the R&D team thought about testing
our proteins using an SDS-Page technique in order to isolate and see our protein of interest.

Many SDS-Pages were performed in order to characterize our 7 sequences one by one. But
first bibliographical research are needed to find the molecular weight of our part sequences:

* PHAC1: between 48 and 63 kDa [1]
* AmyH: 50.189 kDa [2]
* PCT: 67 kDa [3]
* EL222: 24.741 kDa [4]
* LDH: 37.049 kDa [5]
* PlAase (RPA1511): 34, 1 kDa [6]
* mtrC: 71.237 [7]

![Electrophoresis Gel made by the technique of SDS-Page](https://static.igem.wiki/teams/4187/wiki/results/results-img-19.png)

**Figure 19: Electrophoresis Gel made by the technique of SDS-Page.**
From the left to the right the sequence loaded in wells are : 2x BL21 NT (control) (1-2), 2x EL222 (3-4), 2x
AmyH (5-6), Molecular weight (7), 3x PCT (8-10), 3x PHAC1(11-13).

![SDS-PAGE and profile of the PageRuler Plus Prestained Protein Ladder](https://static.igem.wiki/teams/4187/wiki/results/results-img-20.png)

**Figure 20: SDS-PAGE and profile of the PageRuler Plus Prestained Protein Ladder from ThermoFisher.**

As you can see on the Figure 19, the first control of *E.Coli BL21 wt* in position 1 worked well
since no strip is bigger than the others in the first well. We will use this well as a reference
(negative control) to compare to others Electrophoresis Gel.

When we are comparing the Figure 19 with the Figure 20, we can admit that the 4th well is
corresponding to the part named *EL222* since one strip is thicker than the others and her
molecular weight is around 24 kDa.

Now we can observe that on all the wells corresponding to PCT (from number 8 to 10), a
thicker strip just below the strip of the molecular weight corresponds to 70 kDA (Figure 20).

Even if our electrophoresis gel is kind of blurry with the first test (Figure 19) we can
assess that *BL21 wt* worked well as a negative control. And looking at the characterization of
our part; *EL222* is expressed in our construction as well as *PCT* since the proteins of interest
produced are over-expressed in the electrophoresis as we can see on the Figure 19 at the
corresponding Molecular Weight.

![Electrophoresis Gel made by the technique of SDS-Page](https://static.igem.wiki/teams/4187/wiki/results/results-img-21.png)

**Figure 21: Electrophoresis Gel made by the technique of SDS-Page.**
From the left to the
right: Molecular Weight (1), PCT (2), mTRC (3), PHAC1 (4), AMYH (5), Molecular Weight
(6), BL21 wt (7), PLAase (8), EL222 (9), PHAC1 (10).

As we can observe on this second gel the *BL21 wt*, our negative control worked well since
no strips are overexpressed in the 7th well.

When we compare the 7th well and the molecular weight in the 6th position a thicker strip is
seen in the 4th well corresponding to the loading of the transformed bacteria with *PHAC1*. In
fact the thicker band is between the molecular weight of 55 kDa and 70 kDa (Figure 20), and
corresponds to the expected molecular weight of *PHAC1* which is between 48 kDa and 63
kDa.

Moreover, on the 5th well corresponding to *AMYH* we can observe a thicker strip between
the molecular weight (6) of 35 kDa and 55 kDa (Figure 20), that is around 50kDa which
correspond to the molecular weight of the expressed protein *AMYH* leading to the validation
of our engineering success.

![Electrophoresis Gel made by the technique of SDS-Page](https://static.igem.wiki/teams/4187/wiki/results/results-img-22.png)

**Figure 22: Electrophoresis Gel made by the technique of SDS-Page.**
From the left to the
right: Molecular Weight (1), BL21 wt (2), AMYH (3), mTRC (4), LDH (5), Molecular Weight
(6), PCT (7), EL222 (8), PHAC1(9), PLAase (10).

On the fifth well, we observed a band slightly above 55 kDa that could correspond to
LDH because its molecular weight of the expressed LDH is at 57 kDa. (see Figure 20) We
know that *E.Coli* expresses its own Lactate dehydrogenase which has a Molecular weight
around 36kDa [8]. On the first gel, we observe the same bands as here that explains why.
We can suppose that our LDH is expressed with the support of its characterisation in the
Proof of Concept part

As a conclusion on the SDS Page performed by the team, we succeed in proving the
expression of most of our parts such as *AMYH, EL222, PHAC1, LDH* and *PCT*. This may
validate the right construction of our sequences. But other sequences such as *PLAase* and
*mTRC* still need to be characterized on another electrophoresis gel.

Since *mTRC* was transformed into *Shewanella oneidensis*, the wet lab team should
have loaded a “*Shewanella oneidensis wt*” as a negative control in the electrophoresis gel to
properly interpret the mTRC expression or not, but due to a lack of time we didn’t perform it
yet.

## References

1. Matsumoto, K., Tobitani, K., Aoki, S., Song, Y., Ooi, T., & Taguchi, S. (2014). Improved
   production of poly(lactic acid)-like polyester based on metabolite analysis to address the
   rate-limiting step. *AMB Express*, 4(1), 83.
2. UniProt, 2022. Q9L4I9 · Q9L4I9\_9GAMM. Available at:
   <uniprot.org/uniprotkb/Q9L4I9/entry#sequences> (accessed on 10/08/2022)
3. Selmer, T., Willanzheimer, A. and Hetzel, M. (2002), Propionate CoA-transferase from
   *Clostridium propionicum*. European Journal of Biochemistry, 269: 372-380.
   <https://doi.org/10.1046/j.0014-2956.2001.02659.x>
4. UniProt, 2022. Q2NB98 · *LVHTH\_ERYLH*. Available at:
   <https://www.uniprot.org/uniprotkb/Q2NB98/entry> (accessed on 10/08/2022)
5. UniProt, 2022. P26297 · *LDHD\_LACDA*. Available at:
   <https://www.uniprot.org/uniprotkb/P26297/entry> (accessed on 10/08/2022)
6. Hajighasemi M, Nocek BP, Tchigvintsev A, Brown G, Flick R, Xu X, Cui H, Hai T,
   Joachimiak A, Golyshin PN, Savchenko A, Edwards EA, Yakunin AF. Biochemical and
   Structural Insights into Enzymatic Depolymerization of Polylactic Acid and Other Polyesters
   by Microbial Carboxylesterases. Biomacromolecules. 2016 Jun 13;17(6):2027-39.
7. UniProt, 2022. *Q8EG34 · Q8EG34\_SHEON*. Available at:
   <https://www.uniprot.org/uniprotkb/Q8EG34/entry> (accessed on 10/08/2022)
8. UniProt, 2022. *P52643 · LDHD\_ECOLI*. Available at:
   <https://www.uniprot.org/uniprotkb/P52643/entry> (accessed on 10/08/2022)

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