iGEM 2019 — Team Orleans

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# Team:Orleans/Notebook

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# Notebook

2019

july

22 july- 28 july

Reception of our Bacteria, Cupriabidus Metallidurans CH34 and Escherichia Coli S17-1, the growth (on plate and liquid culture) for glycerol stock.
After receiving the Cupriavidus Metallidurans CH34 and Escherichia Coli S17-1 bacteria, the received bacterial pellets were suspended in BM3 and subsequently placed in a continuous culture of these two strains.
Glycerol stocks are the realized to preserve these strains.
These bacteria were also spread on Petri dishes to ensure that they were free of contamination and that the cells were viable.
First determination of Metals in station sludge by ICP for Pb and Cd in supernatant and dry matter.
The various sludge samples received from the wastewater treatment plant, including Tamburg sludge (T) and excess sludge (BE), were analysed by ICP to determine their cadmium and lead concentration.
For each sludge, a sample was dried in the oven at 105°C over night and a supernatant sample after settling was taken.
All these samples are acidified by adding 6mL of HNO3 to 65% and 3mL of HCl to 37%.
The dried samples are mineralized by heating in a microwave oven, then all samples are filtered on 0.22µm cellulose membranes.
These samples are then measured by ICP.
Determination of sludge dryness
The dryness of the different samples of sewage sludge was determined by weighing for each sludge a sample before and after drying in an oven at 105°C over night.
The calculation of the dryness is as follows:

**Results for the dryness calculation :**

![](https://static.igem.org/mediawiki/2019/6/65/T--Orleans--siccitetab.png)

**Results of dryness :**

![](https://static.igem.org/mediawiki/2019/e/ef/T--Orleans--siccitegraph.png)

august

29 july - 4 august

Receipt of bacteria
C. Metallidurans was cultured to amplify it and perform genomic DNA purification using the genejet genomic DNA purification kit.
PCR on C. Metallidurans genome
PCR amplification on the metallidurans genome to amplify the sequences bordering the targeted genes in 3' using the primers designed in June.
Solutions prepared for PCR (50µL):
10 µL Tp Phusion HF 5X
5 µL dNTP
5 µL Fwd primer
5 µL Rev primer
2.5 µL Plasmid (1ng/mL, 0.1ng/mL or 0.01ng/mL)
22 µL H2O (QSP)
0.5 µL DNA pol Phusion
Purification of PCR products by the genejet gel extraction kit and DNA cleanup micro kit

![](https://static.igem.org/mediawiki/2019/c/ca/T--Orleans--Photo1.png)

Migration of PCR products to agarose gel
These gels show the correct functioning of all the PCRs of the 3' inserts.
Purification by Plasmidic Miniprep of plasmids pSB1C3 and double digestion by the restriction enzymes EcoR1 and Spe1
Continuation of 3' PCR (CzcD and CzcA genes)

![](https://static.igem.org/mediawiki/2019/6/69/T--Orleans--Photo2.jpg)

Digestion of Biobrick in pSB1C3 after amplification in DH5alpha and miniprep of this plasmid

19 august - 25 august

![](https://static.igem.org/mediawiki/2019/6/61/T--Orleans--Photo4.jpg
)

PCR amplification of 5' inserts

Receipt of plasmids pCM184 and pBBR1MCS-2 from Addgen and amplification in DH5α
Miniprep plasmid of these plasmids then migration on gel
Here the PCR Amplifications of the 3' inserts all worked, however, only the 5' inserts of the pbrR and pbrT genes and CzcA were correctly amplified

![](https://static.igem.org/mediawiki/2019/8/8e/T--Orleans--Photo5.jpg)

Double digestion pBBR1MCS-2 and pCM184 from DH5α.

![](https://static.igem.org/mediawiki/2019/f/f2/T--Orleans--Photo3.jpg)

Purification by plasmid Miniprep of pSB1C3, double digestion by EcoRI and SpeI of the biobrick and purification on gel

![](https://static.igem.org/mediawiki/2019/e/e1/T--Orleans--gel_photo_6.jpeg)

Purification by plasmid Miniprep, double digestion (by the same restriction enzymes) and purification on gel of digested pBBR1MCS-2

![](https://static.igem.org/mediawiki/2019/1/16/T--Orleans--gel_photo_7.jpeg)

PCR on Colony of construction pCM184 - insert 3'

![](https://static.igem.org/mediawiki/2019/d/d4/T--Orleans--gel_photo_8.jpg)

Digestion of plasmid pCM184 and 5' inserts (pbrR, pbrT, czcA) and Ligation in pCM184 Then Transformation S17-1

![](https://static.igem.org/mediawiki/2019/0/0c/T--Orleans--gel_photo_9.jpg)

PCR on colonies from previous transformations

26 august - 1 september

![](https://static.igem.org/mediawiki/2019/d/d7/T--Orleans--gel_photo_10.jpg)

Digestion of the plasmid pBBR1MCS-2 with the restriction enzymes SpeI and EcoRI
Digestion by the same restriction enzymes and purification of biobrick on gel
Transformation of S17.1 by the construction pBBR1MCS-2 + Biobrick

september

2 september - 8 september

![](https://static.igem.org/mediawiki/2019/8/8c/T--Orleans--Gel11.jpeg)

PCR on colonies from the previous transformation

![](https://static.igem.org/mediawiki/2019/0/0e/T--Orleans--Gel12.jpeg)

Double digestion by the restriction enzymes BamHI and EcoRI and Pan ligation in pBBR1MCS-2 + Biobrick
Addition of the Pan developer to the construction pBBR1MCS-2 - Biobrick
It was digested by the restriction enzymes HindIII and EcoR1.
Similarly, the construction pBBR1MCS-2 - Biobrick was purified by the GeneJET Plasmid Miniprep Kit, and digested by the same restriction enzymes as Pan
The digested promoter was then linked to the construction digested by the T4 ligase.

![](https://static.igem.org/mediawiki/2019/3/37/T--Orleans--Gel13.jpeg)

Migration of the construction pBBR1MCS-2 + Biobrick + pan before and after digestion by EcoRI
The ligation product was then introduced by transformation into E. Coli S17.1 to amplify it.

9 september - 15 september

Colony PCR on the last transformation
The colonies obtained were then tested by PCR on colonies to select the bacteria transformed with the new construction pBBR1MCS-2 - Pan - Biobrick.

16 september - 22 september

![](https://static.igem.org/mediawiki/2019/a/a0/T--Orleans--Gel14.jpeg)

colony PCR on the biobrick after electroporation (failed) on C. Metallidurans CH34

30 september - 6 october

![](https://static.igem.org/mediawiki/2019/b/bf/T--Orleans--Gel15.jpeg)

second electroporation + colonie miniprep (2 negatives / 2 positifs)

![](https://static.igem.org/mediawiki/2019/f/ff/T--Orleans--Gel16.jpeg)

Realization of the final gel showing the different constructions realized


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# Team:Orleans/Experiments

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close

# Experiments

[Overview](#design-part1-section)[Heavy metal resistance modification](#design-part2-section)[Ethanol](#design-part3-section)[Initial and final tests](#design-part4-section)[References](#references-section)

Summary

[Initial test](#design-part1-section)

[Heavy metal resistance modification](#design-part2-section)

[Ethanol metabolsim implementation](#design-part3-section)

[Final tests](#design-part4-section)

[Primer table](#design-part5-section)

[Medium](#design-part6-section)

[References](#references-section)

Inital tests

Goal

This section aims to do all of the tests and measurements necessary before beginning to work with our biological and non-biological material.

Principle

The ability of bacteria to grow on the different medium and the absence of contamination is determined by solid and liquid culture of each strain on the different growth medium. Concentration of compounds of interest in sludges are also measured by using appropriate technics. Heavy metal concentrations are measured by ICP, glucose by DNS methode and ethanol, by densimeter.

Material & methods

**Strain growth**

Cupriavudus metallidurans CH34 and E. coli S17-1 are plated on Browth Medium 3 (BM3) and 284 Medium then incubated for 1day (E. coli) or three days (C. metalidurans). the number of colonies on the plates is then counted. Bacteria are the inculated in BM3 and 284, and the OD660 is measured at t=0, t=1 and t=3 days. For both plate and liquid culture, a negative control without bacteria is also carried out.Stock glycerols have also been produced to preserve the strains used.

optical density measurementclick to view experimental protocol

Sample collection:
Take 200µL of culture medium (white) or bacterial culture and place them in a spectrometric tank.
Add 800mL of water
Reading of the optical density:
Read the optical density at 600 nm using a spectrophotometer

cell aliquot storageclick to view experimental protocol

Final glycerol concentration is recommended to be 15%, so for a final volume of 1 mL it is necessary to add 250 µL of stock glycerol 60%
1. Put 750 µl of the cell culture into an Eppendorf tube and add 250 µl of 60% Glycerol. Vortex the mix.
2. Store in the -80ºC freezer

**Sludge drying :**

Sludges is consisted of water and dry matter. Dryness is the mass percentage of dry matter.
Dryness is assessed by the amount of solid remaining after heating to 105°C over night. It is expressed as a percentage by weight.
The dryness is dertermined by the the following equation :

![](https://static.igem.org/mediawiki/2019/3/36/T--Orleans--formulesiccite.png)

**Sludge heavy metal concentration measurement**

Sewage sludges are dryed at 105°C. 200mg of dried sudge is put in specific acid volume for the mineralisation. Finally the samples are dilutes in QSP 50 ml of distilled water before vaccum filtration. The quantity of heavy metal is measured by ICP then reported to the dry mass of sludge.

ICP measurementsclick to view experimental protocol

Mineralization :
Dry the samples in an oven (100°C).
Take 200mg of dry matter and put them in teflon tubes.
Add 6mL of HNO3 and 3mL of HCL to each sample tube.
Place the tubes in the digestion chamber and carry out a heating cycle (heating up to 160°C for 15 minutes, then stabilize at this temperature for 40 minutes, and finally the cooling phase at 55°C for 15 minutes).
The samples are then placed in 50mL tubes (QSP 50mL H2O).
Filter the samples using a vacuum pump and nitrocellulose filters (0.45µm pore).
Store in a cold room until the samples pass through the ICP.

ICP (Inductively Coupled Plasma Spectrometry):
Place 5mL of each previously mineralized sample in the machine.
Start the machine with standard range with a mixed solution of lead and cadmium.
Make the measurement and report the data on the dry mass of sludge.

Heavy metal resistance modification

Goal

The main objective is to strategically remove heavy metal resistance genes of C. Metallidurans to specialize it or improve their aggregation of heavy metals.
More precise:

* Improve aggregation of Cadmium by removing czcD gene
* Decline aggregation of Cadmium (and specialize itself for others heavy metals) by removing czcA gene
* Decline aggregation of Lead (and specialize itself for others heavy metals) by removing PbrR and/or PbrT

Only the PbrR gene deletion could be performed later.

Principle

The principle is to use the vector pCM184 with target gene’s borders to replace it, by homologous recombination, with a selection gene (Kanamycine resistance), then see if the gene deletion has an effect on resistance of others heavy metals.
For more details, see Design.

Material & methods

**Target gene borders PCR amplification :**

A genenomic purification (using the GeneJET Genomic purification Kit from Thermofisher Scientific) is realized on a Cupriavidus metallidurans CH34 Culture.
Each borders are amplify by PCR, using a pair of specific primers (see primers table)

**Plasmid construction for homologous recombination using pCM184 :**

pCM184 (from addgene) plasmid and upstream target gene border are both degested by BglII and NdeI restriction FastDigest enzymes (thermofisher scientific) then - after a verification of the digestion success by electrophoresis- the insert is ligated into pCM184 and the resulting plasmid is transformed and amplificated in Escherichia coli DH5alpha by chemical transformation. To check the ligation success, a colony PCR is realized over plasmid minipreparation (using the GeneJET Plasmid Miniprep Kit from Thermofisher Scientific) from the transformed E. coli which are positive to the colony PCR. These manipulations are repeated for downstream target gene border to obtain the functionnal Homologeous recombination plasmid.

E. coli transformationclick to view experimental protocol

Preparations of competent bacteria (for freezing)

The day before, take a colony of E. coli, inoculate 2 to 5ml of LB and incubate at 37°C overnight while shaking. Autoclave 100ml of LB (500ml erlen)

1. Take 1ml from the preculture to inoculate the 100ml of LB.
2. Incubate at 37°C (stirring), and check the DO600 (from 2 to 4 hours depending on the strains)
3. When the DO600 reaches 0.5, centrifuge the culture in 3 sterile 50ml Falcons (5 min, 4000 g, 4°C). Remove the supernatant.
4. Gently resuspend the bacterial pellets in 30ml (10ml per tube) of cold TFB1, collect the resuspensions in 1 tube, and leave the tube in the ice for 1h30.
5. Centrifuge the tube (5min, 4000g, 4°C) and remove the supernatant.
6. Resuspend gently in 3ml of cold TFB2, and aliquot in sterile microtubes (100 µl).
7. Freeze the microtubes at -80°C

Processing of competent bacteria (frozen)
1. Thaw the competent bacteria on the ice (50µl of bacteria = 1 transformation).
2. Switch on the water bath at 42°C.
3. Add to the 50µl of competent bacteria (in the ice) the plasmid to be transformed (previously cooled in the ice):
- 50 to 100 ng of pure plasmid
- 5 µl of ligation product
4. Mix gently by turning and leave in the ice for 20 minutes
5. Put the microtubes at 42°C for exactly 90 seconds.
6. Put the tubes back in the ice for 2-3 minutes, then add 450µl of LB
7. Grow at 37°C by shaking the tubes for 45min at 1h
8. Spread on a box (LB-Agar + antibiotic) with glass beads
- 50µl if transformation by pure plasmid
- 200 to 250µl if processed by ligation product
9. Leave the boxes (agar at the top) in the oven at 37°C for one night.

Tampon TBF1 :
100mM RbCl, 50mM MnCl2, 30mM KAcO, 10mM CaCl2, 15% glycerol
Tamponner à pH 5,8
Filtrer sur 0.22µ - garder à 4°C

Tampon TBF2 :
10mM MOPS, 10mM RbCl, 75mM CaCl2, 15% glycerol
Tamponner à pH 6,8
Filtrer sur 0.22µ - garder à 4°C

colony PCRclick to view experimental protocol

Preparation of the solution:
In PCR tubes make the following solution:
- 10 µL of green taq master mix kit (thermo scientific)
- 6 µL of water
- 2 µL of Forward primer (10mM)
- 2 µL of Reverse primer (10mM)
Colony sampling:
Sterile, remove a colony transformed with a cone.
Soak this cone in the previously made colony PCR solution.
Place this cone in a sterile culture tube and store it in the refrigerator.
Revelation of positive colonies:
Place the PCR tubes on colony in a thermal cycler and use the program provided by the green taq master mix kit (thermo scientific)
Then migrate the PCR product (agarose gel 1%) by electrophoresis
Select the positive colonies according to the electrophoresis strips (of the expected size) and add 5mL of culture medium with antibiotic to the culture tubes containing the corresponding colonies collected.

**Homologous recombination**

To achieve the transformation of Cupriavidus metallidurans CH34 by the plasmid containing the homology sequences to the genes targeted for homologous recombination, the conjugation transfer method was performed. The Cupriavidus metallidurans CH34 strain and Escherichia coli S17-1 bearing the pCM184 derivated plasmid are both grown over-night in BM3 at 30°C. Bacterias are then, whased and concentrated (by centrifugation) in NaCl 0.9% three times. The cell pellet is mixed in even quantities then after 5 minutes, a drop of the mix is dropped on a BM3 plate then incubated for 5 days at 30°C. the bacteria thus obtained are resuspended in 0.9% NaCl, shaked for 10 minutes, then inoculated at different concentration in 284 medium with kanamycin for selection of C. metallidurans recombinants.

**Mutants functionnality tests**

Recombinants and wild type C. metallidurans are inoculated into 284 medium and grown for 2 days. A negative control is realized without inoculation (equivalent volume of sterile milliQ mater is added). A t=0, t=1day and t=2days, a sample is collected in each tube. bacteria are separated from the remaining and the concentration in heavy metal in both bacterial and non-bacterial fractions are measured by ICP. The bacteria quantity in each tube is evaluated by plating a sample on BM3.

Ethanol metabolism implementation

Goal

The objective is to provide to our C. metallidurans strain, alcoholic fermentation genes (pyruvate decarboxylase and pyruvate deshydrogenase of Zymomonas mobilis) to permit it to produce ethanol from glucose.
To do this, we use the fusion protein of BBa\_K1122673 part ([Edingurgh 2013 team](http://parts.igem.org/Part%3ABBa_K1122673)) under Pan promoter control (ref parts : pan + composite), to obtain strong expression, in particular in presence of heavy metals.
We have made an expression plasmid construction by inserting in vector pBBR2MCS-1, for expression at C. Metallidurans, the Pan promoter and the fusion protein ORF of the part BBa\_K1122673.
Then, C. Metallidurans is transformed with this construction by electroporation.

Principle

On construit un plasmide d'expression en insérant dans le vecteur d'expression pBBR2MCS-1 pour une expression chez métallidurans le promoteur pan et l'ORF de la protéine fusion XXX. On transforme ensuite CH34 par ce plamide par éléctroporation.

Material & methods

**Expression vector construction**

the pBBR1MCS-2is and the BBa\_K1122673 part are digested by EcoR1 and Spa1 Fast digest restriction enzymes (Thermofisher Scientific). After purification by GeneJET Gel Extraction and DNA Cleanup Micro Kit (Thermo fisher) and checking the digestion success by electrophoresis, the BBa\_K1122673 ORF insert is ligated into pBBR1MCS-2 and the resulting plasmid is transformed into Escherichia coli DH5alpha. The ligation success is verified by realizing a colony PCR. The Pan promoter is digested and inserted into pBBRMCS- BBa\_K1122673 by following the same process than for BBa\_K1122673, with HindIII and EcoR1 Fast digest restriction enzymes (Thermofisher Scientific).

**CH34 electroporation transformation**

C. Metallidurans CH34 is grown overnight in BM3, then diluted in a bigger volume of BM3 and re-incubated at 30°C for 2 days. Cells are then cooled in ice for 20minutes, centrifugated (15 minutes at 4000g) and resuspended into washing buffer (compsition - see table). the centrifugation and resuspension in washing buffer is repeated 3 times. The last re-suspension volume is calculated to obtain an optical density of 120 at 660nm. the bacteria are aliquoted and 4µL of the plasmid solution (100ng/4µL) are added to the samples to be measured. Samples are incubated 1 minute in ice before being transferred in cooled electroporator cuve and placed into the electroporator. After the electroporation, 1ml SOC medium is added immediately to the bacterias. Suspension is transferred in tubes and incubated at 30°C for 1h, then plated on selective medium ().

**Functionality test on recombinant cells for the ethanol production**

To get cellulose, cleaning paper is blended in water. First, a digestion test of the paper mixed by cellulases from Trichoderma reesei is performed by adding 125 U of cellulases to 300mg of paper and incubated for 24 hours at 37°C. The results are observed through the disappearance of paper aggregates.
Then, 1,2g of mixed paper is added to 250mL of 284 medium in presence of cellulase. The Other conditions are the direct presence of glucose in medium 284 (instead of gluconate), a rich medium (BM3), and autoclaved or non-supplemented sewage sludge.
pBBR1MCS-2-pan- BBa\_K1122673 transformed C. metallidurans or CH34 are inoculated in the different conditions (using a BM3 preculture) in presence of cellulase or not, then the glucose and ethanol concentrations are measured from T=0h at T=26h by using DNS method for the glucose and densimeter method for the ethanol. Negatives control without bacteria is also carried out.

DNS assayclick to view experimental protocol

Prepare the following DNS solution:
- 3,5-DNS 1% : 10g/L
- NaOH 0.4M : 16g/L
- Double tartrate (Na;K).4H2O 30%: 300g/L
Heat the solution to dissolve it at 90°C in a bain-mari.
Adjust the volume after heating with water.
Realization of a calibration range:
Achieve a glucose concentration range of 0 to 10 mM (including the expected concentration range in the samples).
Add to 100µL of each glucose solution of known concentration, 200µL of DNS solution.
Heat to 95°C for exactly 5 minutes.
Allow to cool and then deposit 200µL of each sample on a 96-well plate.
Sample production:
For each sample, add 600µL of DNS solution to 300µL of sample and heat to 95°C for exactly 5 minutes.
Allow samples to return to room temperature.
Place 200µL of each sample in triplicate in the 96-well plate.
Determination of glucose concentration:
Read the absorbance at 530nm of the standard range and of each sample (in triplicates).
Make a calibration curve of the absorbance as a function of the known glucose concentrations.
Report the absorbance values at 530nm of the samples on the calibration curve and deduce their glucose concentration

Final tests

Goal

The final tests confirmed the functionality of the project, that is to say, that Cupriavidus metalidurans has been modified by homologous recombination and is able to better or more specifically remove heavy metals from sludge, and that it is able to produce ethanol in sludge from glucose, obtained by digesting cellulose naturally present in sludge by cellulases.
Comparisons were made between C. metallidurans delaPbrR, CH34 and E. Coli S17-1 (negative control) strains for their heavy metal depollution capacity.
A comparison was also made between cultures grown in autoclaved or non-autoclaved sludge.
For these tests, several types of cultures are carried out.
The three strains are first put in the presence of Lead and Cadmium, in order to activate the resistance mechanisms to heavy metals.
These three strains are then cultivated in autoclaved or non-autoclaved sludge for 20 hours at 30°C.
An ICP analysis is performed, before and after culture, under the different culture conditions as well as on the bacteria alone (separated from the sludge).

Principle

Heavy metal depolluting test on the different strains cells : The three strains C. metalidurans CH34, C. metalidurans ΔPbrR and E. Coli S17-1 are initially seeded in BM3 and incubated at 30°C for 2 days to achieve preculture.
Each strain is then centrifuged and resuspended in medium 284 supplemented with lead (0.4mM) and cadmium (2mM) and incubated for 15 mins at 30°C.
This incubation allows the activation and implementation of the resistance mechanisms of C. metalidurans, so that they are already expressed during the culture in the sludge.
The concentrations of these metals have been chosen in such a way that they are just below the lethal concentrations.
After activation, the three strains are centrifuged and then resuspended under two culture conditions. The first is a culture in non-autoclaved sewage sludge, supplemented with BM3 (50/50, v/v). The second condition is a culture in autoclaved sewage sludge, supplemented with BM3 (50/50, v/v).
Growth monitoring was carried out in each culture condition, by measuring optical density at 600nm and spreading diluted samples by a factor of 102 and 106 on solid BM3 medium in order to count the bacteria.
After 20 hours of incubation, the different cultures were centrifuged, first at 300g for 5 minutes to recover the solid matter without bacteria contained in the sludge, then at 14000g for 5 minutes to recover the bacteria from each condition.
The solid matter, the bacterial pellet and the supernatant from the 14000g centrifugation were used to perform an analysis of the quantities of lead and cadmium by ICP.
The same manipulations were also carried out on mud samples supplemented with BM3, which were not sown with bacteria.

Material & methods

The three strains C. metalidurans CH34, C. metalidurans ΔPbrR and E. Coli S17-1 are initially seeded in BM3 and incubated at 30°C for 2 days to achieve preculture.
Each strain is then centrifuged and resuspended in medium 284 supplemented with lead (0.4mM) and cadmium (2mM) and incubated for 15 mins at 30°C.
This incubation allows the activation and implementation of the resistance mechanisms of C. metalidurans, so that they are already expressed during the culture in the sludge.
The concentrations of these metals have been chosen in such a way that they are just below the lethal concentrations.
After activation, the three strains are centrifuged and then resuspended under two culture conditions. The first is a culture in non-autoclaved sewage sludge, supplemented with BM3 (50/50, v/v). The second condition is a culture in autoclaved sewage sludge, supplemented with BM3 (50/50, v/v).
Growth monitoring was carried out in each culture condition, by measuring optical density at 600nm and spreading diluted samples by a factor of 102 and 106 on solid BM3 medium in order to count the bacteria.
After 20 hours of incubation, the different cultures were centrifuged, first at 300g for 5 minutes to recover the solid matter without bacteria contained in the sludge, then at 14000g for 5 minutes to recover the bacteria from each condition.
The solid matter, the bacterial pellet and the supernatant from the 14000g centrifugation were used to perform an analysis of the quantities of lead and cadmium by ICP.
The same manipulations were also carried out on mud samples supplemented with BM3, which were not sown with bacteria.

Primer table

Here are documented all the primer pairs we used in our experiments.
In the table, each pair is composed of two successive primers. the forward primer (first one in the table order) have is name ended by "\_fwd" as the reverse primer has a name ended by "\_rev".
The 8 first pairs correspond to the primer used for Cupriavidus metallidurans CH34 gene border amplification in perspective of homologous recombination. For all of these plasmids name is composed as follow :
"target gene name - side of the gene -(5 for upstream and 3 for downstream) \_ orientation of the primer (forward or reverse)". The primer whom the name begins by Col is used in colony PCR, and the couple Cre157\_fwd - Cre157\_rev is designed to amplify Cre fragment from pCM157.

name

sequence

size

CzcD-5\_fwd

AAAAGATCTGGTGCTGATGACGGCCCTGG

29nt

CzcD-5\_rev

AAACATATGCCTGCGCCCATAGAAACTCC

29nt

CzcD-3\_fwd

AAAGGGCCCCAGGAGGAAACTAAGGTGCG

29nt

CzcD-3\_rev

AAAGTTAACAATCGTCTGCCCCCAACTCG

29nt

PbrR-5\_fwd

AAAAGATCTTAACGGCTAGCGTTCGCTGC

29nt

PbrR-5\_rev

AAACATATGTCATGGCGTCGGATGGGAGA

29nt

PbrR-3\_fwd

AAAGGGCCCCCCATCCAAGCGACTAGGTG

29nt

PbrR-3\_rev

AAAGTTAACCGACGTGGCCATCCTTGACG

29nt

PbrT-5\_fwd

AAAAGATCTATGAGCACATCCGTCAGGTC

29nt

PbrT-5\_rev

AAACATATGGCATGGGAGGCCTTACC

26nt

PbrT-3\_fwd

AAAGGGCCCGGCCATCTACCCAGGTGTAA

29nt

PbrT-3\_rev

AAAGTTAACGGATCTGGCATAACGTGGTG

26nt

CzcA-5\_fwd

AAAAGATCTGGCTTGGCGACCGGGTC

26nt

CzcA-5\_rev

AAACATATGACATTGCTGTTTCCCCCGTA

29nt

CzcA-3\_fwd

AAAGGGCCCAAGGCCGCCAGCCTGC

25nt

CzcA-3\_rev

AAAGTTAACGCCAAATGTCCGCTTCTTGT

29nt

Col-5\_fwd

CATTTCCCCGAAAAGTGCC

19nt

Col-5\_rev

AGATTTTGAGACACAACGTGG

21nt

Col-3\_fwd

ACACCTTCTTCACGAGGCAGAC

22nt

Col-3\_rev

ACAATCACGAAACGCGGCAG

20nt

Col-pBBR\_fwd

GTTGGGTAACGCCAGGGT

18nt

Col-pBBR\_rev

ACACAGGAAACAGCTATGAC

20nt

Col-Cre\_fwd

TTTTCAATATTATTGAAGCATTTATCAGG

29nt

Col-Cre\_rev

GCTTGAGCGTGACAATCACG

20nt

Cre157\_fwd

AAAAGATCTAATACGCAAACCGCCTCTC

28nt

Cre157\_rev

AAAGTTAACCCTCTTCGCTATTACGCCAG

29nt

Mediums

Here are documented all the mediums we used in our experiments.

Lysogeny Broth

Description

Lysogeny broth, abreviated LB, is a common bacterial growth medium. It's particularly suited for *Escherichia coli* growth.

Nutrient broth

Description

Nutrient broth, abreviated NB, is another common bacterial growth medium. its composition is verry similar to the LB one. It's commonly used for *Cupriavidus metallidurans CH34* growth.

284

Description

284 (Mergeay, 1995; Schlegel et al., 1961) is a tris buffered medium supplemented in heavy metal salts. It's used to study bacteria heavy metal resistance. the recipe presented below is from S. Monchy's thesis [284 composition source].

Recipe

284 medium

ingredient

quantity

Tris

50mM

NaCl

80mM

KCl

20mM

NH4Cl

20mM

Na2SO4

3mM

MgCl2 6H2O

1mM

CaCl2 6H2O

0.2mM

Na2HPO4 2H2O

0.4% (v/v)

Fe(III)NH4 citrate

1% (v/v)

solution SL7

0.1% (v/v)

gluconate

0.2% (m/v)

SL7 solution

ingredient

quantity

HCl

10mM

ZnSO4 7H2O

0.5mM

MnCl2 4H2O

0.5mM

H3BO3

1mM

CoCl2 6H2O

0.8mM

CuCl2 2H2O

0.1mM

NiCl2 6H2O

0.1mM

Na2MoO4 2H2O

0.15mM

Super Optimal Broth with Catabolite Expression

Description

Super Optimal Broth with Catabolite Expression, abreviated SOC, is a medium designed to optimize bacterial transformation efficiency. Here, it is used to resuspend bacteria after the electroporation.

References

[conjugaison] Wiesemann N, Mohr J, Grosse C, et al. Influence of copper resistance determinants on gold transformation by Cupriavidus metallidurans strain CH34. J Bacteriol. 2013;195(10):2298–2308. doi:10.1128/JB.01951-12
[pCM184] Broad-host-range cre-lox system for antibiotic marker recycling in gram-negative bacteria. Marx CJ, Lidstrom ME. Biotechniques. 2002 Nov;33(5):1062-7. 10.2144/02335rr01 PubMed 12449384
[pCM157] Broad-host-range cre-lox system for antibiotic marker recycling in gram-negative bacteria. Marx CJ, Lidstrom ME. Biotechniques. 2002 Nov;33(5):1062-7. 10.2144/02335rr01 PubMed 12449384
[electroporation] Taghavi S, van der Lelie D, Mergeay M. Electroporation of Alcaligenes eutrophus with (mega) plasmids and genomic DNA fragments. Appl Environ Microbiol. 1994;60(10):3585–3591.
[pBBR1MCS-2] Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA, Roop RM 2nd, Peterson KM. Gene. 1995 Dec 1;166(1):175-6. 0378111995005841 [pii] PubMed 8529885
[biobrick] http://parts.igem.org/wiki/index.php?title=Part:BBa\_K1122674
[pan] Ribeiro-dos-Santos, G., Biondo, R., Quadros, O. de F., Vicente, E. J., & Schenberg, A. C. G. (2010). A metal-repressed promoter from gram-positive Bacillus subtilis is highly active and metal-induced in gram-negative Cupriavidus metallidurans. Biotechnology and Bioengineering, 107(3), 469–477. https://doi.org/10.1002/bit.22820
[composition du millieu 284] Sebastien MONCHY, "Organisation et expression des gènes de résistance aux métaux lourds chez Cupriavidus métallidurans CH34", These de doctorat en Sciences de la vie sous la dirrection de Max MERGEAY et Ariane TOUSSAINT, Bruxelles, Uinversité Libre de Bruxelles, 2007, 99p

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# Results

[overview](#design-part1-section)[heavy metal resistance modification](#design-part2-section)[ethanol](#design-part3-section)[test initiaux et finaux](#design-part4-section)[references](#references-section)

Summary

[Part 1](#design-part1-section)

[Part 2](#design-part2-section)

[Part 3](#design-part3-section)

[Part 4](#design-part4-section)

[Part 5](#design-part5-section)

[References](#reference-section)

Part 1 : Construction of model S17.1 - pCM184 - 5' -3' inserts

The principle here is to provide a plasmid construct to the wild type strain of C. metallidurans CH34, which will allow, in the presence of a recombinase already expressed in it, to perform a homologous recombination of a targeted gene, with a sequence provided in this construct.
This sequence then includes two homology sequences of 300 bp each, in 5' and 3' borders of the target gene, as well as the Kanamycin resistance gene in the centre, in order to select the bacteria in which the deletion will have taken place.
The strain of E. Coli S17.1, was used and transformed with the construction since it has the ability to conjugate with C. metallidurans, in order to transfer the plasmid it contains to it, without the need for a helper plasmid.
The plasmid pCM184, chosen here as the basic vector for the plasmid construction, contains an OriT sequence, allowing the plasmid to be transferred from S17.1 to C. Metallidurans, and the gene expressing oriT recognising protein.
It also contains the Kanamycin resistance gene, with an MCS upstream and downstream of it.
It is then sufficient to add the two homology inserts 5' upstream and 3' downstream of kanamycin on this plasmid.
The four targeted genes are pbrR, pbrT, czcA and czcD. (They have function for metal resistance)
Thus, sequences present in the 5' and 3' borders of each of these genes were selected in order to amplify them by PCR on the genome of C. metallidurans.
These PCR products were then digested by restriction enzymes, in the same way as the plasmid pCM184, in order to insert the 3' inserts into it by ligation, first, then the 5' inserts in a second step.

![](https://static.igem.org/mediawiki/2019/e/e9/T--Orleans--gelfinal1.png)

An agarose gel was used to summarize all the plasmid constructions obtained (Final Gel 1).
This one, makes it possible to prove the presence of the 5' and 3' inserts of 300pb each, by the size of the strip corresponding to the construction in its linear form.
Indeed, since the plasmid alone has a size of 6760 bp, it is possible to differentiate the plasmid containing only the 3' insert (size 7022 bp), or containing both 5' and 3' inserts, in its linear form (7299 bp).
Similarly, these vectors were digested by the two restriction enzymes used during their construction, in order to highlight the presence of each of the inserts.
Due to the malfunction of the ligation step for some constructs, only the pbrR gene was targeted for deletion in C. metallidurans.
Thus, the conjugation was performed between strain S17.1 - pCM164 - inserts pbrR 5'-3' and C. metallidurans CH34 wild type.
These two strains were then placed in direct contact on a solid medium and incubated for 5 days at 30°C.
After conjugation, the colonies of C. metallidurans transformed and selected by the presence of Kanamycin, were isolated in order to recultivate them and perform PCR on the genome, in order to prove the absence of the pbrR gene (size 438 bp), replaced by the Kanamycin resistance gene (size 816 bp).
Amplification was therefore carried out from primers hybridizing to the two homology sequences 5' and 3', in order to obtain a size of 1416 bp on gel if the homologous recombination took place, or a size of 1038 bp if it did not take place.
The gel results for the pbrR gene then showed that the construction of the C. metallidurans strain ΔpbrR was successful.

Part 2 : Construction of model S17.1 - BioBrick+Pan

To enable ethanol production to be provided mainly from glucose, present in sludge or produced by the degradation of cellulose, a fusion protein adhB - pdc is provided to C. metallidurans, to enable the conversion of pyruvate into ethanol.
The aim is then to provide glucose by digesting the cellulose present in the sludge, in order to provide an additional source of carbon to produce ethanol, without impacting the growth of bacteria.
For this purpose, the BBa\_K1122673 share was used to optimize this conversion of pyruvate to ethanol.
The part was digested by the restriction enzymes EcoRI and SpeI, in order to insert it into the plasmid pBBR1MCS-2. This plasmid was used because of its replication capacity in both E. Coli and C. metallidurans, by the presence of the replication origin oriV.
Similarly, this plasmid contains an MCS in the vicinity of the Lac promoter, allowing induction of the expression of a gene provided by IPTG.

![](https://static.igem.org/mediawiki/2019/5/5a/T--Orleans--Gel_SDS_1.png)

SDS Gel 1 : Expression test of the fusion protein adhB - pdc from BBa\_K1122673 part (noted BB) by IPTG induction in E. Coli S17.1 and under the Pan promoter control (noted Pan) in C, metallidurans CH34 on SDS page gel

Thus, this construction pBBR1MCS-2 - fusion was introduced by transformation into S17.1, in order to perform a first expression test of this fusion protein by induction with IPTG (SDS gel 1).
This induction test, has been migrated to SDS - Page gel, in order to highlight the expression of the fusion protein.
From the sequence of this fusion protein, it is possible to theoretically calculate the molecular weight of this protein, which is then 100.92 kDa.
On the gel, a band appears and intensifies with induction time at about 100 kDa, corresponding well to the expected results.
Then, a strong constitutive promoter in E. Coli and C. metallidurans (Pan promoter) was added to the construction, so as to express at least as strongly the fusion protein as with the Lac promoter in S17.1 and constitutively.
To do this, the promoter and the plasmid construct were digested by the restriction enzymes BamHI and EcoRI, so that the promoter was inserted by ligation upstream of the fusion protein gene.
This new construction pBBR1MCS-2 - Pan - fusion, is then introduced in S17.1, to first test the expression of the fusion protein and compare it to induction by IPTG.

![](https://static.igem.org/mediawiki/2019/0/0e/T--Orleans--Gel_SDS_2.png)

SDS Gel 2 : Expression test of the fusion protein adhB - pdc from BBa\_K1122673 part (noted BB) by IPTG induction in E. Coli S17.1 and under the Pan promoter control (noted Pan) in C. metallidurans CH34 or on C. Metallidurans with pbrR gene deletion (noted ΔpbrR) in different culture conditions and with or without activation on 284 medium supplemented by lead and cadmium durring 15 minutes (noted activated), on SDS page gel

The results on SDS - Page gel show an expression approximately equal to that of induction by IPTG after 20 hours of incubation. (SDS Gel 2)
These results show that this promoter allows the strong and constitutive expression of the fusion protein in E. Coli S17.1.
This plasmid construction was then introduced into C. metallidurans by electroporation.
All the steps in the implementation of this pBBR1MCS-2 construction containing the Pan promoter, followed by the fusion protein, were demonstrated by electrophoresis. (Final Gel 2)

![](https://static.igem.org/mediawiki/2019/4/45/T--Orleans--gelfinal2.png)

Similarly, the expression of the fusion protein, under the control of the Pan promoter in C. Metallidurans, was shown by SDS - Page gel and compared to the induction expressions in IPTG or under the control of the Pan promoter, in E. Coli S17.1. gel (SDS 1 and 2)
The results of the SDS - Page gel thus show a similar expression in C. Metallidurans, and in S17.1 (induction with IPTG or under the control of the Pan promoter).
This then proves that the fusion protein is well expressed in C. Metallidurans and in a strong and constitutive way.

Part 3 : Proof of concept

Cellulose

Before performing the sludge field tests, we performed various tests under medium conditions 284 (heavy metal supplemented media) in which we added different sources of sugars to create minimum media in order to demonstrate that our bacteria can consume glucose.
We prepared a first medium 284+Glucose as a control and a medium 284+paper to reproduce a glucose source similar to sludge. The paper is first digested by a mix of cellulase from Trichoderma reesei.
Glucose consumption capacity will first be assessed by measuring the OD of the cells growth.

![](https://static.igem.org/mediawiki/2019/f/fa/T--Orleans--suivi_DO_DNS_284_glucose.png)

OD measurment during time of bacteria in 284 Glucose medium

For medium 284 Glucose (Figure 284 Glucose), we observe an increase in OD, which indicates that bacteria can use it to grow. However, it should be noted that growth appears to be slightly higher for the bacterium Δpb than for the CH34+BB\_pan strain.
In a second step, the same measurements are made in medium 284 Paper (284 Paper), here again, bacterial growth can be observed.

![](https://static.igem.org/mediawiki/2019/e/ea/T--Orleans--suivi_DO_DNS_284_paper.png)

OD measurment during time of bacteria in 284 Paper medium

The latter is again more important for the bacteria Δpb than for the strain CH34+BB\_pan.
In parallel, an assessment of the amount of glucose in the medium was made via a DNS assay. In both cultures, there is a decrease in the glucose level in the environment, which shows that it is indeed consumed by the bacteria CH34+BB\_pan and by Δpb.

![](https://static.igem.org/mediawiki/2019/2/29/T--Orleans--glucose_rate_284gluc.png)

The bioavailable glucose level in medium 284 Glucose (Figure Glucose rate in 284 Glucose medium) is fixed and more important at the base. Glucose consumption also seems similar for the two strains CH34+BB\_pan and Δpb. In contrast to the glucose level in condition 284 Paper (Figure Glucose rate in 284 Glucose Paper). Indeed, in the latter, cellulase continues to digest cellulose and there is therefore a part of the consumption that is compensated by this "production" of glucose.

![]( https://static.igem.org/mediawiki/2019/8/84/T--Orleans--glucose_rate_284pap.png)

Nevertheless, the high rate of decrease after 24 hours shows a higher sugar consumption in this condition. It was also found that CH34+BBB\_pan appears to consume less glucose than Δpb, which corresponds to the slower growth of this modified strain of metallidurans. The large size of the BioBrick and the very strong promoter that precedes it force the bacteria to produce the protein of interest, which consequently leads to a decrease in its division rate and therefore a lower glucose consumption due to the lower number of bacteria.
It can therefore be conceptually assumed that the modified bacteria are capable of growing in sludge because they are able to use cellulose from paper digested via cellulase.

Heavy metals

To verify the effectiveness of our bacterial construction on metals, we carried out a first bacterial culture in medium 284 supplements (284 supp) in Lead (0.5mM) and Cadmium (2mM). These concentrations were not taken at random, the lead concentration corresponds to the minimum growth inhibitory value (MIC) of C. Metallidurans CH34 and that of Cadmium is less than half of this MIC and still activates the mechanisms of Cadmium resistance.
The 3 bacterial strains tested, E. Coli S17-1, C. Metallidurans CH34, and C. Metallidurans ΔpbrR, were pre-cultured for 36 hours in Broth Medium 3 containing no heavy metals and then cultured for 24 hours in the tested medium (284 supp).
An Optical Density follow-up was performed to evaluate the potential inhibition of C. Metallidurans ΔpbrR by Lead.
Finally, the different cultures were stopped and centrifuged to recover the bacterial pellet and supernatants separately.
The 2 samples were determined for lead and cadmium by ICP, and the pH was measured in the supernatants.
**Optical Density Tracking**

![](https://static.igem.org/mediawiki/2019/5/50/T--Orleans--OD_284.png)

According to the follow-up of the Optical Density in 284, ΔpbrR and S17-1 saw their growth limited in 21 hours of cultivation. On the other hand, the results show exponential growth from CH34 to 22 hours of cultivation.
**pH results**

![](https://static.igem.org/mediawiki/2019/6/63/T--Orleans--ph284.png)

The pH measurement allows visualizing the impact of the bacterial strain on the pH of the medium.
It should be noted that since the solubility limit for lead is 7 when the pH is acid below this value, it becomes more and more soluble. On the other hand, when the value is basic, lead tends to create aggregates.
We can notice that strain S17-1, significantly acidifies its environment thus facilitating the solubilization of lead and thus an increase in its concentration in the supernatant of the media. On the other hand, C. Metallidurans CH34 is alkaline in its media, which must induce aggregation of the lead in the medium and therefore the decrease of the concentration in the supernatant.
**Lead Assay at ICP**

![](https://static.igem.org/mediawiki/2019/6/62/T--Orleans--LeadMass284_Dry.png)

Lead assay at ICP indicates a lower concentration in the supernatant for bacterial strains S17-1 and CH34, near or lower concentrations than control, indicating for S17-1 solubilization of lead bound to acidity. medium, and an efficient CH34 aggregation mechanism reducing the amount of lead in solution.
On the other hand, ΔpbrR which no longer has a lead efflux mechanism has a richer lead environment.

![](https://static.igem.org/mediawiki/2019/b/b3/T--Orleans--LeadMass284_Pellet.png)

Here we can find a small amount of lead retained by bacteria or aggregated in S17-1 because the bacterium does not have a strong system of aggregation of lead unlike CH34 and also has an acidic medium.
For the bacterial strain CH34, the amount of lead in the bacterial pellet may very well reflect the aggregation of a pH-related lead coupled with aggregation related to the resistance mechanisms of the bacterium.
Finally, for ΔpbrR, the concentration in the bacterial pellet must certainly be related to the alkaline pH of the medium and therefore to the aggregation of lead, accentuated by the absence of resistance and efflux system of the modified strain.
**Cadmium assay at ICP**

![](https://static.igem.org/mediawiki/2019/6/67/T--Orleans--CadMass284_Dry.png)

For cadmium, we can see here the lowest concentration in CH34 indicating an efficient membrane aggregation system.

![](https://static.igem.org/mediawiki/2019/0/06/T--Orleans--CadMass284_Pellet.png)

Here, the results indicate a good aggregation of cadmium in ΔpbrR and CH34, thus an efficient but weaker aggregation mechanism. This slight difference is certainly due to the difference between bacteria CH34 and ΔpbrR.

Part 4 : Construction of the model ΔpbrR - Pan - fusion protein

By the same method as during the construction of the CH34 - Pan - fusion protein strain, the ΔpbrR strain was transformed by electroporation in order to provide it with the expression construction of the fusion protein.
Thus, this new strain has been tested for the expression of the fusion protein under different culture conditions.
This strain was grown in BM3 medium, in a minimal medium of 284 supplemented with lead and cadmium and presenting only glucose as a carbon source, as well as in medium 284 containing cellulose and in the presence of cellulases in order to degrade it into glucose and other reducing sugars.
The expression of the fusion protein is observed after 20 hours of incubation at 30°C, by SDS - Page gel (SDS Gel 3).

![](https://static.igem.org/mediawiki/2019/4/45/T--Orleans--gelfinal2.png)

It is then possible to see a slightly variable expression between the different conditions.
The expected results were a stronger expression in the presence of Cadmium, due to the promoter Pan.(1)
Indeed, since bacterial concentrations here are relatively low, these differences in expression could not really be observed.

Part 5 : Application in sludge

Cellulose

After proof of concept, the same tests as before are performed in autoclaved sludge (AB) to test our bacterium under real application conditions.
First, OD monitoring is performed in sludge containing cellulase or not containing cellulase (Figure Autoclaved Sewage).

![](https://static.igem.org/mediawiki/2019/3/35/T--Orleans--suivi_DO_DNS_BA.png)

A similar bacterial growth of Δpb+BB\_pan bacteria with or without cellulase is observed. This allows us to see that cellulase has no effect on the growth of our bacteria.
In a second step, the DNS monitoring made it possible to monitor the consumption of glucose in the sludge autoclaved by the bacteria.

![](https://static.igem.org/mediawiki/2019/b/be/T--Orleans--glucose_rate_BA.png)

A decrease in the available glucose concentration can be observed under both cellulase and non-cellulase conditions (Figure Glucose rate in Autoclaved Sewage). This corresponds to the results found in Part 3. First, it can be noted that glucose consumption is higher in the cellulase-free condition despite bacterial growth in both conditions. This can be explained on the one hand because the autoclave has released sugars that bacteria can use to develop... but the condition with cellulase allows to degrade more of them, which buffers the loss related to bacterial development.
All these experiments have therefore enabled us to demonstrate the survival of our modified C. metallidurans in a glucose-rich environment and the use of this sugar in the metabolism of the bacteria. Our model has also succeeded in operating in real conditions in autoclaved sludge.
From the SDS gels pages, we also showed that our bacterium produced the fusion protein through the implementation of BioBrick.
So all that remained was to measure the ethanol produced to complete our results... Unfortunately, due to many problems, we were unable to achieve them on time. We ordered a dosage kit in June that never arrived, so we found a plan B using a densitometer (which is usually used to measure the alcohol content of wine). Unfortunately, it was not sensitive enough. Finally, we had the idea to carry out a colorimetric assay using alcohol dehydrogenase, but we were unable to implement it due to a lack of time.

Heavy metals

**Followed by Optical Density in the Purification Sludge**

![](https://static.igem.org/mediawiki/2019/e/e9/T--Orleans--ODSewagesludge.png)

According to the monitoring of Optical Density, bacterial growth is performed uniformly in sludge.
**Determination of pH in sewage sludge**

![](https://static.igem.org/mediawiki/2019/c/c1/T--Orleans--phSew.png)

The pH values observed on the cultures in the sludge of purification are all in basic conditions. On the other hand, we can see slight acidification of the medium by S17-1 as in the proof of concept in the medium 284supp.
**Determination of lead at ICP in sludge**

![](https://static.igem.org/mediawiki/2019/d/d9/T--Orleans--LeadMassSew_Dry.png)

The results here demonstrate a high concentration of lead in the dry matter for ΔpbrR and CH34. This certainly demonstrates a strong aggregation of lead due to the mechanisms or the alkaline pH of the medium.

![](https://static.igem.org/mediawiki/2019/b/b1/T--Orleans--LeadMassSew_Super.png)

The concentration values in the supernatants appear much lower than in the dry matter. Nevertheless, a slightly lower concentration can be observed for strain CH34 and a higher concentration for S17-1.

![](https://static.igem.org/mediawiki/2019/7/72/T--Orleans--LeadMassSew_Pellet.png)

Finally, in the bacterial pellet, we can see a higher concentration of lead attached to the bacteria of the genus CH34 and equivalent results in S17-1 and ΔpbrR. This demonstrates the loss of the lead resistance function in the modified ΔpbrR strain.
**Determination of Cadmium in sewage sludge**

![](https://static.igem.org/mediawiki/2019/1/11/T--Orleans--CadMassSew_Dry.png)

Cadmium concentrations are higher in the dry matter of CH34 and ΔpbrR. These results could be evidence of aggregation by CzcABC systems of its two strains, inducing sedimentation at 300RCF Cadmium, although this remains to be verified in the following results.

![](https://static.igem.org/mediawiki/2019/b/bb/T--Orleans--CadMassSew_Super.png)

Here the solution concentration values in the supernatants are substantially the same for the 3 strains.

![](https://static.igem.org/mediawiki/2019/d/d9/T--Orleans--CadMassSew_Pellet.png)

Here we can see that cadmium concentrations in bacterial pellets are very low. This indicates that membrane aggregation systems are very inefficient in CH34 and ΔpbrR. We can explain this by the concentration in the sludge of purification too weak to allow the activation of its last ones.

Reference

1. A metal-repressed promoter from gram-positive Bacillus subtilis is highly active and metal-induced in gram-negative Cupriavidus metallidurans. - PubMed - NCBI. https://www.ncbi.nlm.nih.gov/pubmed/20517979.