iGEM 2019 — Team Lund

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# Team:Lund/Notebook

iGEM Lund 2019

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# Lab Notebook

Whenever one works on a project, it's important to keep track of what has been done. This page contain a week by week recollection of what we've done in the lab. This text was transcribed from a physical notebook used by the lab group.

### Week 1 (8-14 July)

*E coli* Nissle 1917 identification:

Colony PCR was conducted on colonies extracted from Mutaflor® -pills. The PCR product taken from pMUT2 was faulty (no bands appeared at the expected 313 bp), and it was concluded that the pMUT2 plasmid wasn’t present in our bacteria. Two sets of new primers were designed and ordered.

Colony specific PCR was conducted with the correct primers, and a band was discovered at ~400 bp - EcN was isolated.

A trial of PCR was conducted for amplification of the ordered genblocks as they hadn’t arrived yet.

Julia, Mateusz, Savvina, Manjyot

### Week 2 (15-21 July)

The gene-blocks arrived. They were resuspended and gradient amplification PCR and agarose gel-electrophoresis were conducted. The DNA was then purified according to the kit protocol. PCR conditions were optimal for all but the lead T7 gene-block, and different temperatures and mastermix conditions were tested until optimal conditions were found.

Julia, Mateusz, Savvina, Manjyot

### Week 3 (22-28 July)

The ends of our geneblocks and pUC19 was digested with restriction enzymes before running electrophoresis and isolating the correct genes. Ars-Tac, Ars-T7, Pb-T7 were found and purified with a purification kit. Before ligation, we precipitated the DNA-Vector mix into separate solutions. Two ratios of DNA vector were used for ligation, (Vector:DNA) 1:1 and 3:1. OD of ligated product was measured. We transformed the ligated mix with heat-shock into TG1 *E. coli* and plated over the weekend.

Julia, Mateusz, Savvina, Manjyot

### Week 4 (29 July- 4 August)

Monday morning came, no growth of cells were shown. We realised that our digestion protocol was incorrectly performed. We mixed two different buffers.

We started over with a double digestion, each buffers separately, with the restriction enzymes PcsI and NdeI.

PCR amplification and gel electrophoresis was run.

The gBlocks MBL and Ars-Tac, Ars-T7, Pb-T7, were purified and ligation was continued..

The gBlocks were then transformed into TG1. PCR amplification and gel electrophoresis was run.

cPCR was run to confirm transformation.

Julia and Mateusz

### Week 5 (5-11 August)

The rest of the crew return along with them a readily ligated ArsR-T7 plasmid arrived. The plasmid was resuspended to a final concentration of 25 ng/ μL. Transformation of the ArsR-T7 plasmid was conducted into TG1, *E. coli* Nissle and *E. coli BL21-DE3.* Colony PCR was then conducted to confirm transformation. Agarose gel electrophoresis was run of the colony PCR.

Cultivation was made with Ars-T7 in *E. coli* Nissle and control of *E. coli* Nissle. The cultivation was done in triplicates. The monitored parameters were of pH, OD, and protein expression (with SDS-PAGE and Bradford assay).

Julia, Mateusz, Savvina, Manjyot

### Week 6 (12-18 August)

The same plasmid, ArsT7, was then transformed using electroporation into *E. coli* BL21-DE3.

Colony PCR was then conducted to confirm transformation.

Cultivation was made with Ars-T7 in *E. coli* BL21-DE3 and control of *E. coli* BL21-DE3. Different induction times were tested in this cultivation: OD600nm = 0.5 & 1.2. The monitored parameters were of pH, OD, and protein expression (with SDS-PAGE and Bradford assay).

Julia, Mateusz, Savvina, Manjyot

### Week 7 (19-25 August)

Staining and destaining of SDS-PAGE gel.

Already ligated MBL-ArsR-T7 and Pb-T7-plasmids arrived. Transformation of the plasmids was achieved into *E. coli* BL21-DE3using heat shock*.* Colony PCR was conducted to confirm transformation. Results of the colony PCR were inconclusive and therefore single point digestion was done and then run on an agarose gel electrophoresis. The gBlock Pb-Tac was digested using double digestions. The pUC19 was then phosphorylated and the gBlock was ligated. The ligase was inactivated and the plasmid was transformed into *E.coli* TG1 cells. Colony PCR was conducted to confirm transformation of these cells, alongside the previously mentioned *E.coli* BL21-DE3 containing MBL-ArsRT7 and PbT7 with different annealing temperatures. Then gel electrophoresis was conducted.

Cultivation was made with Ars-T7, MBL-ArsRT7 and Pb-T7 in *E. coli* BL21-DE3, and control of *E. coli* BL21-DE3. During the cultivation different induction times were tested OD600nm = 1 , 2.5, 3.5 & 6 h. The monitored parameters were of pH, OD, and protein expression (with SDS-PAGE and Bradford assay). Check the induction from the SDS

Julia, Mateusz, Savvina, Manjyot

### Week 8 (26-1 August- September)

Staining and destaining of SDS-PAGE gel was conducted.

Digestion of Pb-Tac, run agarose gel and continue with purification using a kit.

The plasmids was ligated and then transformation in *E. coli* TG1.

Julia, Mateusz, Savvina, Manjyot

### Week 9 (2-8 September)

The plasmid from the registry, BBa\_J04450 was also transformed in *E. coli* TG1, *E. coli* Nissle and *E. coli* BL21-DE3*.* These were cultivated during 24 hours, parameters measured were OD and fluorescence.

Julia, Mateusz, Savvina, Manjyot

### Week 10 (9-15 September)

Colony PCR was done to confirm transformation. The desired transformation of cells was conducted, followed by gel electrophoresis. No clear bands were visible. Pre-culture was prepared in 5 mL LB.

Electrocompetent cells *E. coli* Nissle were prepared. Glycerol stock was also prepared.

Cultivation was made with Pb-Tac in *E. coli* Nissle and control of non transformed *E. coli* Nissle. The monitored parameters were , OD and protein expression (with SDS-PAGE).

Julia, Mateusz, Savvina, Manjyot

### Week 11 (16-22 September)

Staining and destaining of SDS-PAGE gel.

Cultivation was made with Ars-Tac ,Pb-Tac in *E. coli* Nissle alongside a control of non-transformed *E. coli* Nissle. After overnight cultivation the OD was normalized at 2h and the cultures were harvested and resuspended in new media containing different concentrations of toxic metals.The monitored parameters were OD, and toxic metal concentration which were analyzed externally.

Julia, Mateusz, Savvina, Manjyot

### Week 12 (23-29 September)

Staining and destaining of the SDS-PAGE gel.

The plasmid from the registry, BBa\_J04450 was also transformed in *E. coli* TG1, *E. coli* Nissle and *E. coli BL21-DE3. T*hese were cultivated during 24 hours, parameters measured were OD and fluorescence.

Cultivation was made with Ars-T7 ,Pb-T7 in *E. coli* BL21-DE3 and control of *E. coli* BL21-DE3 as well as Ars-Tac ,Pb-Tac in *E. coli* Nissle and control of *E. coli* Nissle. The pH in the culture was adjusted to a lower value in order to mimic conditions similar to the digestion process. After overnight cultivation, the OD was normalized at 2h and the cultures were harvested and resuspended in new media containing different concentrations of toxic metals. The monitored parameters were of pH, OD, and toxic metal concentration which were analyzed externally.

Julia, Mateusz, Savvina, Manjyot

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# Team:Lund/Experiments

iGEM Lund 2019

* [Home](/Team%3ALund)
* Project

  + [Description](/Team%3ALund/Description)
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  + [Model](/Team%3ALund/Model)
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[Blog](/Team%3ALund/Blog)

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# Protocols and Experiments

## Protocols

### PCR (Amplification of gBlocks)

#### Mastermix:

 For each 20 µL reaction mixture, mix the following:

* 2 µL dNTP mix
* 2 µL Forward primer
* 2 µL Reverse primer
* 0.1 µL Phusion High Fidelity DNA Polymerase
* 2 µL Phusion buffer
* 10 µL MilliQ water

#### Thermal Cycler Settings:

1. 98°C for 180 seconds
2. 98°C for 20 seconds
3. Set a gradient for 55.9°C, 57.1°C and 62.1°C for 60 seconds
4. 72°C for 90 seconds
5. Repeat step 1-4 for 25 cycles
6. 72°C for 10 minutes
7. Store at 4C° until loading

### Gel Electrophoresis

#### Gel preparation:

1. Weigh 1.5 gram agarose.
2. Mix in 100 mL TAE buffer.
3. Warm the mixture in microwave and heat until the solution is clear.
4. Add 12 µL GelRed after the solution has cooled down.
5. Fix comb and add the solution into the casket for the gel to solidify.

#### Ladder & samples:

1. Mix 2 µL gene ruler with 2 µL loading dye and 1 µL MilliQ water for preparation of ladder.
2. Mix 10 µL of PCR samples with 2 µL loading dye.

#### Electrophoresis conditions:

1. Remove the comb and move the solidified gel to the electrophoresis set up.
2. Fill the chamber with TAE buffer until all the wells are covered completely.
3. Load gel and run it for 90 minutes at 100 V.

### Colony PCR

#### Template preparation

1. Pick colonies with inoculation loop and re-suspend in 50 μL of MilliQ water.

#### PCR reaction mixture

For each 20 µL reaction mixture, mix the following:

* 2 µL dNTP mix
* 2 µL Forward primer
* 2 µL Reverse primer
* 0.1 µL Dream Taq DNA Polymerase
* 2 µL Dream Taq buffer
* 2 µL template prepared in previous step
* 10 µL MilliQ water

#### Thermal Cycler Protocol

1. 95°C for 5 minutes
2. 95°C for 30 seconds
3. 59°C for 30 seconds
4. 72°C for 45 seconds
5. Repeat above steps for 30 cycles
6. 72°C for 5 minutes
7. Store at 4°C until loading

### Transformations

 For TG1 and BL21-DE3 cells (Heat Shock):

1. Add 2 μL of plasmid DNA to 50 μL cells on ice (no more than 5 μL, i.e. no more than 10% volume of cells).
2. Swirl gently to mix the contents.
3. Incubate on ice for 30 minutes.
4. Transfer tubes to shaking incubator at 42oC for 90 seconds.
5. Place samples back on ice and let them cool for 1-2 minutes.
6. Add 800 μL of LB media.
7. Incubate for 60 min in the shaking incubator at 37°C, for the cells to revive.
8. Plate 75-100  μL of the culture on LB agar and incubate overnight at 37 °C.

 For *E. coli Nissle* cells (Electroporation):

1. Keep 2 mm electroporation cuvettes on ice to cool.
2. Let the electrocompetent EcN cells (-80°C) thaw completely on ice.
3. Add 1 μL of pure plasmid DNA to 25 μL of electrocompetent EcN cells.
4. Pipet the mixture into chilled cuvette and flick to deposit cells at the bottom.
5. Electroporate at 1800 V for 5 ms.
6. Add 975 μL of LB medium to the cuvette, and pipette up and down to resuspend the cells.
7. Transfer to a culture tube and place in shaking incubator for 1 hour at 37 °C.
8. Plate 75-100  μL of the culture on LB agar and incubate overnight at 37 °C.

### Medium Preparation

#### LB Media (for 1 L):

* 5 g Yeast Extract
* 10 g Tryptone
* 10 g NaCl
* Weigh ingredients individually and put them in a schott bottle with a magnetic stirrer
* Fill with 1 L of distilled water
* Stir until all powder is diluted

#### TB Media (for 1 L):

* 12 g Peptone
* 24 g Yeast Extract
* 4 mL Glycerol
* 2.3 g KH2PO4
* 12.5 g K2HPO4
* Weigh and later autoclave Potassium salts, glycerol separately and peptone along with yeast extract to avoid precipitation
* Dissolve all the components in a sufficient amount of distilled water but not exceeding the total 1 L volume

#### Enriched Complex Media (for 1 L):

* 20 g Peptone
* 20 g Yeast Extract
* 30 g Glucose
* 13 g KH2PO4
* 10 g K2HPO4
* 5 g NaCl
* 2 g (NH4)2SO4
* 1 g MgSO4 \* 7 H2O
* 0.2 g NH4Cl
* Weigh and later autoclave Potassium salts,  Sulfate salts, Chloride salts, glucose separately and peptone along with yeast extract to avoid precipitation
* Dissolve all the components in a sufficient amount of distilled water but not exceeding the total 1 L volume

#### LB Agar ( for 1 L  approximately 45- 50 Petri Dishes):

* 5 g Yeast Extract
* 10 g Tryptone
* 10 g NaCl
* 150 g agarose
* Weigh ingredients individually and put them in a schott bottle with a magnetic stirrer
* Fill with 1 L of distilled water
* After autoclaving, cool the media and add 100 μg/ml of antibiotic of choice (ampicillin, carbenicillin, chloramphenicol) depending on the application
* Pour into sterile Petri dishes

### SDS-PAGE

#### Reagents:

1. Make 50 mM sodium acetate buffer at pH 5.0
2. Pre-casted gel (Bio-Rad)
3. Loading buffer (sample buffer):
   1. 3.3 ml of TRIS 0.5 M pH 6.8
   2. 8 ml of SDS 10% w/v 25
   3. 4 ml of glycerol
   4. 1 ml of bromophenol blue 0.2% w/v
   5. 2.62 g of DTT (dithiotreithol)
   6. Adjust volume to 20 ml using MQ-water- Electrophoresis (Running) buffer:
     1. 7.5 g of Tris
     2. 36 g of glycine
     3. 2.5 g of SDS
     4. Adjust pH to 8.3 using HCl and make the final volume up to 500 ml using MQ-water. Dilute the buffer 5 times
   - Molecular weight marker- Staining solution:
       1. 1 g of Coomassie 0.2%
       2. 200 ml of MeOH 40%
       3. 50 ml of HAc 10%
       4. 260 ml of MQ-water
     - De-staining solution:
       1. 40% methanol
       2. 10% acetic acid
       3. 50% water

#### Sample Preparation:

1. Check OD and normalize all the samples to OD 2.
2. Resuspend pellet in 20 µl of 2x SDS loading dye.
3. Boil samples at 95°C for 10 min.
4. Load the prepared samples into the gel (Bio Rad plates, 15 well of 15μL, 4 - 20% gradient gel).
5. After loading, fill the apparatus with the buffer solution between the glass plate until it reaches the maximum volume.

#### Running the Gel:

1. Run the gel at 80 V until the dye front migrates into the running gel (~10 min).
2. Increase to 160 V until the dye front reaches the bottom of the gel (~30 min).

#### Staining & Destaining the Gel:

1. Remove the gel from the apparatus.
2. Remove the spacers and glass plates, by using the metal spatula.
3. Place the gel in a plastic container.
4. Add ~20 ml staining solution ( 50% methanol, 10% HAc & 0.1% Coomassie Brilliant Blue) and stain for 1 hour in a shaking plate.
5. Pour off the staining solution back to the Schott bottle.
6. Add ~5 mL destaining solution ( 50% Methanol & 10% HAc) and destain briefly  for ~1 min.
7. Pour off  the destaining solution in the active carbon containing Schott bottle.
8. Add ~ 10 mL of destaining solution.
9. Destain in a shaking plate until the gel is visibly destained (overnight).
10. Pour off  the destain solution in the active carbon containing Schott bottle.
11. Rinse with DDI H2O.

### Plamid Isolation

 The protocol according to [Nucleospin plasmid kit](https://static.igem.org/mediawiki/2019/9/9d/T--Lund--Plasmid_Isolation_Kit.pdf) was followed for Isolation of plasmid with low copy number.

### DNA Purification The protocol according to [Nucleospin Gel and PCR clean-up kit](https://static.igem.org/mediawiki/2019/3/37/T--Lund--PCR_%26_Gel_clean-up.pdf) was followed for DNA purification. Restriction 1. Premix the following in an eppendorf: * MilliQ water-15 µL * 10 X Buffer Tango- 2 µL * Plasmid to be digested- 1 µL 2. Mix 2 µL of PscI enzyme to the mixture and keep the eppendorf on ice at all times. 3. Mix gently and spin down for a few seconds. 4. Incubate at 37°C for 2 hours. 5. Inactivate PscI by incubating at 65 °C for 20 min. 6. Cool the mixture by keeping on ice. 7. Add 2.5 µL of 10X buffer Tango to the same eppendorf. 8. Add 2.5 µL of enzyme NdeI and vortex briefly. 9. Incubate at 37 °C for 2 hours. 10. Inactivate NdeI by incubating at 65 °C for 20 min. Ligation 1. Mix digested gBlock and plasmid in ratio of 1:1, i.e., 50 ng of gBlock and plasmid, or in the ratio of 1:3, i.e., 60 ng gBlock and 20 ng plasmid. 2. Add 2 µL of T4 DNA ligase buffer. 3. Add 1 µL of T4 DNA ligase. 4. Add MilliQ water to make the volume upto 20 µL. Toxic Metals stock solution Regarding toxic metals Arsenic Standard for ICM-MS and Lead Standard for AAS were purchased from Sigma- Aldrich. For arsenic, in 250 mL culture: * For 200μΜ add 7.5 ml * For 50μΜ add 0.99 ml * For 10μΜ add 0.188 ml For lead, in 250 mL culture: * For 200μΜ add 10.36 ml * For 50μΜ add 2.59 ml * For 10μΜ add 0.52 ml Sample pretreatment for testing (Eurofins ICP-MS) 1. Centrifuge 15 μL of sample at 8000 rpm for 8 minutes. 2. Transfer supernatant to new tubes. pH regulation via buffer 1. Prepare 2 M Na2HPO4 and 1 M citric acid buffer. 2. To obtain pH 3, add 3.42 mL of 2 M Na2HPO4 and 13.25 mL of 1 M citric acid to 250 mL of media. 3. To obtain pH 5, add 8.22 mL of 2 M Na2HPO4 and 8.45 mL of 1 M citric acid to 250 mL of media. BradfordMaterials: * Bovine Serum Albumin (BSA) (Sigma-Aldrich) * Coomassie Brilliant Blue G-250 (Sigma-Aldrich, catalog number: [27815](http://www.bio-thing.com/p868) ) * Methanol * Phosphoric acid (H3PO4) * Bradford reagent: 1. Dissolve 50 mg of Coomassie Brilliant Blue G-250 in 50 ml of methanol and add 100 ml 85% (w/v) phosphoric acid (H3PO4). 2. Add the acid solution mixture slowly into 850 ml of H2O and let the dye dissolve completely. 3. Filter to remove the precipitates just before use. 4. Store in a dark bottle at 4 °C. Prepare Calibration curve: 1. Prepare a 5-point calibration curve with bovine serum albumin (BSA) as standard. 2. Make solutions of 0.2, 0.4, 0.6, 0.8 and 1.0 mg/ml BSA. 3. Rest samples at room temperature and wait exactly 5 minutes. 4. Measure the absorbance spectrophotometrically at 595 nm. Sample handling: 1. Mix 25 µl sample (BSA standard solution or your sample) with 1.0 ml of Bradford reagent. 2. Rest samples at room temperature and wait exactly 5 minutes and then measure the absorbance spectrophotometrically at 595 nm, absorbance will increase over time. Sample preparation for sequencing: 1. Add 5μL of DNA 100 ng/μL. 2. Add 5μL of each primer concentration 5 μM. 3. Mix by inverting 6-8 times in an Eppendorf. 4. Forward and reverse primer should be in different tubes along with 5 μL of DNA in each. Experiments The first parts of experiments were the cloning and isolation of plasmids. The gBlocks with the appropriate biobricks were purchased from IDT and Eurofins. They were amplified using PCR. The PCR product was then inserted into a pUC19 vector using plasmid digestion and ligation. The plasmids were transformed into the cloning strain of *E.coli* TG1.  Some plasmids were purchased as a complete construct with no cloning necessary. Plasmids were isolated and transformed into *E.coli BL21-DE3* cells and *E.coli* Nissle cells. The latter was isolated from the Mutaflor® probiotic supplement. The presence of the proper *E.coli* strain out of the Mutaflor® supplement was verified with cPCR, for this experiment specific primers were designed. For comparison of strains, all experiments were performed using both *E.coli* BL21-DE3 and *E.coli* Nissle*.* The transformed cells were cultivated under differing growth parameters. The parameters that were varied were induction time and pH. Cultivations were performed using shake-flasks in a shake-incubator at 37 °C. The analysis methods used to evaluate the cultivations were spectrophotometric measurements of optical cell density at 600nm, pH-measurements, SDS-PAGE, and Bradford analysis. The genetically engineered cells were tested for toxic metal uptake by cultivating the cells in media containing toxic metals. The concentration of metal in the media was measured at start concentration and continuously during the cultivation to detects changes in concentration. A lower metal concentration in the media would mean uptake of metals into the cells. Before start testing the original construct with *EcN*, preliminary testing of the gene construct took place using *E.coli* BL21(DE3) with T7 promoter. Plasmid purification Five gblocks were purchased from IDT, the gene for ArsR using promoter T7 and Tac, fusion protein of ArsR+ MBL using promoter T7 and genes for PbrD and pbrT using promoters T7 and Tac. Whole plasmids with the genes for PbrD+ pbrT, ArsR and MBL with the T7 promoter was purchased from IDT. An additional purchase from eurofins was made of a whole plasmid with the gene for ArsR using the Tac promoter. The whole plasmids were all delivered in a company specific vector with an ampicillin antibiotic marker. PCR was used to amplify the gblocks, using amplification primers of our own design. The PCR protocol used is described under “Protocols”. The PCR product was run through agarose electrophoresis and purified using a plasmid purification kit found under “protocols”. The purified gblocks were digested according to the digestion protocol found under “protocols”, using the restriction enzymes PscI and NdeI. The vector pUC19 was also digested using the same enzymes. The gblocks and vector was then ligated using T4 ligase while following the ligation protocol under “protocols”. The plasmids were transformed into *E.coli BL21-DE3* chemically competent cells using the heat-shock protocol under “protocols”. Plasmids were transformed into *E.coli Nissle* electrocompetent cells following the protocol for the Nissle electroporation under “protocols”. Cultivation The genetically modified cells were cultivated in LB media in shake-flasks in a shaking-incubator set at 175 rpm and 37 °C. The growth of the cells was analysed and compared to control strain of *E.coli BL21-DE3* and *E.coli Nissle* without the inserted plasmid. The growth was measured using a spectrophotometer to measure the optical cell density at 600nm. Growth curves can be found on the [results page](https://2019.igem.org/Team%3ALund/Results). Two different induction times were tested 0.8 or 2 OD600nm. For the induction Isopropyl β-d-1-thiogalactopyranoside (IPTG) was used.  After induction samples were taken every hour for the monitoring of optical density and prepared for SDS-PAGE. After three constant OD readings the cultivation was terminated, since the cultures had reached stationary phase and the possibility of protein expression was minimized. Testing protein expression Two methods were used to analyse the protein expression; SDS-PAGE and Bradford assay. To analyze the amount of the protein of interest the SDS-PAGE samples were prepared and loaded in the set up as is described in “protocol”. To analyze the total amount of protein a bradford analysis was performed as described above in the “protocol”. Testing of toxic metal uptake to modified cells Metal uptake in the genetically modified cells were tested, both using strain *E.coli BL21-DE3* and *E.coli Nissle.* The cells were cultivated in an enriched complex media, recipe found under protocols. After 12 hours of cell growth, the cells were harvested and put into new media containing different concentrations of the metal. The cells modified to take up arsenic were added to arsenic containing media, and same for the lead uptaking cells.

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# Team:Lund/Results

iGEM Lund 2019

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* Parts

  + [Overview](/Team%3ALund/Parts)
  + [Basic Parts](/Team%3ALund/Basic_Part)
  + [Composite Parts](/Team%3ALund/Composite_Part)
* Practices

  + [Human Practices](/Team%3ALund/Human_Practices)
  + [Public Engagement](/Team%3ALund/Public_Engagement)
  + [SynthEthics](/Team%3ALund/SynthEthics)

[Blog](/Team%3ALund/Blog)

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# Results and Discussion

This is the page were we present our results. This is where we discover if genetically modified gut bacteria can be a part of the solution against exposure to toxic metals.

## Overview

We have successfully sequenced the following parts:

* [BBa\_K3282003](http://parts.igem.org/Part%3ABBa_K3282003) (arsR-T7)
* [BBa\_K3282004](http://parts.igem.org/Part%3ABBa_K3282004) (arsR-Tac)

We have successfully transformed *E.**coli* Nissle 1917 to absorb:

* ~40% of **lead** in a concentration of ~2 mg/L, in 2.5 hours.
* ~20% of **arsenic** in a concentration of ~16 mg/L, in 2.5 hours.

We have failed on the following points:

* To see evidence of protein expression using SDS-page.
* To sequence the parts [BBa\_K3282005](http://parts.igem.org/Part%3ABBa_K3282005) (pbrD-T7) and [BBa\_K3282007](http://parts.igem.org/Part%3ABBa_K3282007) (pbrD-Tac).

## Parts

The following parts were designed:

**Composite part with *arsR* and a T7 promoter: [BBa\_K3282003](http://parts.igem.org/Part%3ABBa_K3282003)**

![](https://static.igem.org/mediawiki/2019/b/bf/T--Lund--arsRTac.svg)

**Composite part with *arsR* and a Tac promoter: [BBa\_K3282004](http://parts.igem.org/Part%3ABBa_K3282004)**

![](https://static.igem.org/mediawiki/2019/2/2b/T--Lund--arsRT7.svg)

**Composite part with *pbrT, pbrD* and a T7 promoter: [BBa\_K3282005](http://parts.igem.org/Part%3ABBa_K3282005)**

![](https://static.igem.org/mediawiki/2019/2/2d/T--Lund--PbT7.svg)

**Composite part with *pbrT, pbrD* and a Tac promoter: [BBa\_K3282007](http://parts.igem.org/Part%3ABBa_K3282007)**

![](https://static.igem.org/mediawiki/2019/a/ad/T--Lund--PbTac.svg)

From now on the parts will be reffered to as the following:

* arsR-T7: [BBa\_K3282003](http://parts.igem.org/Part%3ABBa_K3282003)
* arsR-Tac: [BBa\_K3282004](http://parts.igem.org/Part%3ABBa_K3282004)
* pbrD-T7: [BBa\_K3282005](http://parts.igem.org/Part%3ABBa_K3282005)
* pbrD-Tac: [BBa\_K3282007](http://parts.igem.org/Part%3ABBa_K3282007)

## Isolation of *E. coli* Nissle 1917

*E. coli* Nissle 1917 (EcN) was isolated from a probiotic pharmaceutical Mutaflor. It was resuspended and plated on MacConkey agar that is used to select several *E. coli* strains, among them EcN [1]. In order to confirm the identity of the strain a colony PCR was performed with primers specific for genomic island II (GEI II) and for cryptic plasmid pMUT2 that are specific for EcN. For GEI II the bands were expected at 400bp, while for pMUT2 at 313 bp [2,3]. The results of the colony PCR can be seen in figure 1 below.

![](https://static.igem.org/mediawiki/2019/9/9d/T--Lund--isolation_EcN.png)

Figure 1: Colony PCR result showing well-defined bands with various annealing temperatures, and pMUT2 and GEI II primers.

All colonies picked from MacConkey agar have shown the expected band with GEI II primers but no bands at all with pMUT2. These results confirm the identity of the strain to be EcN, despite negative results for pMUT2 primers since the GEI II primers are specific for chomrosomal region of EcN [2].

## Sequencing of Composite Parts

The following parts were sequenced [ars-T7](http://parts.igem.org/Part%3ABBa_K3282003), [ars-Tac](http://parts.igem.org/Part%3ABBa_K3282004), [pbrD-T7](http://parts.igem.org/Part%3ABBa_K3282005) and [pbrD-Tac](http://parts.igem.org/Part%3ABBa_K3282007).

The sequences of [ars-T7](http://parts.igem.org/Part%3ABBa_K3282003) and [ars-Tac](http://parts.igem.org/Part%3ABBa_K3282004) showed 100% and 99.6% similarity, the alignment results can be found [here](https://static.igem.org/mediawiki/2019/5/52/T--Lund--K3282003-sequencing.txt) and [here](https://static.igem.org/mediawiki/2019/a/ae/T--Lund--K3282004-sequencing.txt) respectively. For [ars-Tac](http://parts.igem.org/Part%3ABBa_K3282004) there are two base substitutions in the sequence. The first one is in the region that is only used for amplification PCR and hence it had no effect on protein structure or expression. However, the second substitution is in the protein coding region and resulted in R18L mutation.

Unfortunately, in the case of [pbrD-T7](http://parts.igem.org/Part%3ABBa_K3282005) and [pbrD-Tac](http://parts.igem.org/Part%3ABBa_K3282007), samples were mixed up or contaminated with [ars-T7](http://parts.igem.org/Part%3ABBa_K3282003) and [ars-Tac](http://parts.igem.org/Part%3ABBa_K3282004) and therefore it was not possible to align the sequences. Due to lack of time it was not possible to repeat sequencing for those composite parts.

## Cultivation of Transformed Bacteria

The *E. coli* BL21(DE3) strain was transformed with biobrick [ars-T7](http://parts.igem.org/Part%3ABBa_K3282003) in pUC19 plasmid by heat shock. Similarly, the BL21(DE3) was transformed with part [pbrD-T7](http://parts.igem.org/Part%3ABBa_K3282005) and EcN strain with part [pbrD-Tac](http://parts.igem.org/Part%3ABBa_K3282007) in pUC19. Unfortunately, *arsR*-Tac arrived late and cultivation could therefore not be performed. The growth rate of the transformed and wild type strains were compared by cultivating the cells in LB media at 37 °C and measuring OD every hour. The cultivation was carried out to investigate if the presence of plasmid acts as a burden for the cells. As is evident from figure 2, the growth rate of the control and the transformed bacteria are very similar. This is unexpected since *arsR* expression has shown to be toxic to cells [4]. This conclusion is a positive indicator that the cells would not die before their intended use of absorbing arsenic.

![](https://static.igem.org/mediawiki/2019/7/73/T--Lund--BL21_arsR-T7.svg)

Figure 2: Comparison of the growth rate of *E. coli* BL21(DE3) transformed with [ars-T7](http://parts.igem.org/Part%3ABBa_K3282003) inserted into pUC19 and wild type *E. coli* BL21(DE3).

![](https://static.igem.org/mediawiki/2019/5/5d/T--Lund--BL21_pbrD_T7.svg)

Figure 3: Comparison of growth rate of E coli BL21(DE3) transformed with [pbrD-T7](http://parts.igem.org/Part%3ABBa_K3282005) inserted into pUC19 and wild type E coli BL21(DE3).

The growth rate of *E. coli* BL21(DE3) containing the lead construct was compared with the wild type BL21(DE3). Figure 3 displays growth of cells over time. The growth rate of the cells containing the construct had a growth rate of 0.474 h-1, and the control of BL21(DE3) cells had a growth rate of 0.263 h-1. The cells containing the constructs grow at a pace that is a bit faster than the control. This result is unexpected, since cells that are forced to express heterologous proteins often have a lower growth rate because of the burden it bears on the cell. The linear fit is also not perfect, with low R2 values compared to what would be wanted. The results could be accidental. However, the results also give an indicator that the cells would not die before they have a chance to perform the desired function of lead uptake.

![](https://static.igem.org/mediawiki/2019/a/ad/T--Lund--EcN_pbrD_Tac.svg)

Figure 4: Comparison of growth rate of EcN transformed with [pbrD-Tac](http://parts.igem.org/Part%3ABBa_K3282007) inserted into pUC19 and wild type EcN.

Figure 4 above shows the growth rate of both transformed and untransformed EcN containing pUC19 with the lead construct. The results are analogous to the *E. coli* BL21(DE3). The growth rate of the EcN containing the plasmid and the control are similar, with the cells containing the construct exhibiting a slightly higher pace. Growth rates of transformed EcN was 0.697 h-1, while the rate for the untransformed control was 0.556 h-1. Since the result is similar to the results in figure 3, so is the discussion. The results could be random from looking at the relatively poor linear fit. The plasmid does from this result not appear to be a burden to the cell and would have a chance to perform the desired function of lead uptake before cell death.

## Toxic Metal Analysis

### Investigation of Composite Parts for Arsenic Accumulation

The part [ars-T7](http://parts.igem.org/Part%3ABBa_K3282003) consists of a T7 promoter ([BBa\_I719005](http://parts.igem.org/Part%3ABBa_I719005)), Anderson RBS ([BBa\_J61109](http://parts.igem.org/Part%3ABBa_J61109)), arsenic resistance operon repressor ([BBa\_K3282000](http://parts.igem.org/Part%3ABBa_K3282000)) and TE terminator ([BBa\_B0012](http://parts.igem.org/Part%3ABBa_B0012)), whereas [ars-Tac](http://parts.igem.org/Part%3ABBa_K3282004) (arsR-Tac)comprises the same basic parts but with a Tac promoter ([BBa\_K3282006](http://parts.igem.org/Part%3ABBa_K3282006)).

The *Escherichia coli* chromosomal *ars* operon confers resistance to trivalent and pentavalent salts of the metalloids arsenic and antimony. *ArsR* is the first gene in the ars operon containing a very specific binding site towards As(III) [4].

#### Experiment

[ars-T7](http://parts.igem.org/Part%3ABBa_K3282003) was inserted into pUC19 and successfully transformed in *E. coli* BL21(DE3), and [ars-Tac](http://parts.igem.org/Part%3ABBa_K3282004) was transformed in the probiotic strain EcN. *arsR* protein expression was demonstrated by performing SDS-PAGE. The cells were cultured in enriched media (containing peptone, yeast extract, phosphates, chlorides and sulphates) at 37°C. They were grown overnight and were harvested in their late exponential phase in order to normalize the OD values to 2. After normalization, the cells were resuspended in fresh enriched medium containing 10 µM, 50 µM and 200 µM of As2O3. As a negative control, the experiment was also performed with wild type *E. coli* BL21(DE3) and wild type EcN in growth medium containing 50 µM As2O3.

#### Results

The SDS-PAGE analysis result can be seen in figure 5 and figure 6. The expected band for *arsR* protein has a molecular weight of 13.5 kDa, therefore it should appear between the two marked points as shown in figure 5 and 6. The gel shows that the engineered bacterial strains, as well as the negative controls have a band with the expected molecular weight. Thus, we can not confirm that it corresponds to *arsR*. It is possible that there are other proteins expressed in *E. coli* of the same molecular weight, masking the expression of *arsR*.

![](https://static.igem.org/mediawiki/2019/3/3e/T--Lund--SDSPAGE_BL21.png)

Figure 5: Samples were harvested at different time points, and the total cellular proteins were analyzed by SDS-PAGE. Protein bands of BL21(DE3) can be seen after induction with IPTG, showing arsR at 13.5 kDa. The control shown is wild type BL21(DE3) without *arsR*.

![](https://static.igem.org/mediawiki/2019/4/46/T--Lund--SDSPAGE_EcN.png)

Figure 6: Samples were harvested at different time points, and the total cellular proteins were analyzed by SDS-PAGE. Image with protein bands of EcN after induction with IPTG, showing ArsR at 13.5 kDa. The control shown is wild EcN without *arsR*.

##### Arsenic accumulation over time

![](https://static.igem.org/mediawiki/2019/b/b6/T--Lund--arsenic_accumulation.svg)

Figure 7: Arsenic concentration in media contaning BL21 transformed with the composite part [ars-T7](http://parts.igem.org/Part%3ABBa_K3282003) or non-transformed *E. coli* BL21 (DE3).

##### Arsenic accumulation over time at varying pH

![](https://static.igem.org/mediawiki/2019/0/0e/T--Lund--arsenic_varying_pH.svg)

Figure 8: Arsenic accumulation over time at varying pH. The blue line represents the arsenite concentration in a culture maintained at pH 3 containing the composite part while the red line represents arsenite concentration in a culture maintained at pH 5 containing the composite part. The yellow line represents the arsenite concentration in a culture with wild type EcN.

Figure 7 and figure 8 compare the arsenite accumulation over time whereas figure 9 and figure 10 compare the growth of engineered as well as wild type strains. It can be seen in figure 7 that the initially added arsenite had a concentration of 3.745 mg/L for both engineered as well as wild type BL21(DE3), and the arsenite concentration measured at 0 hours was 0.014 mg/L and 0.013 mg/L respectively. Similarly, it can be seen in figure 8 that the initially added arsenite had a concentration of 3.745 mg/L ([ars-Tac](http://parts.igem.org/Part%3ABBa_K3282004) at pH3 and Control) and 14.98 mg/L ([ars-Tac](http://parts.igem.org/Part%3ABBa_K3282004) at pH5), and the arsenite concentration measured at 0 hours was 0.012 mg/L, 0.012 mg/L, and 0.024 mg/L respectively. This rapid decrease in both cases was unexpected. The relative concentration of arsenic in media where transformed and non transformed bacteria were present is showcased in figure 11.

##### Growth comparison between transformed and non-transformed *E. coli* BL21(DE3)

![](https://static.igem.org/mediawiki/2019/8/8d/T--Lund--ars_growth_BL21.svg)

Figure 9: Comparison of growth of engineered BL21(DE3) with wild type BL21(DE3) in the presence of arsenic oxide.

##### Growth comparison between transformed and non-transformed EcN

![](https://static.igem.org/mediawiki/2019/0/02/T--Lund--ars_growth_EcN.svg)

Figure 10: Comparison of growth of engineered EcN with wild type EcN in the presence of arsenic oxide.

##### Relative Concentration of Arsenite in Media Containing Transformed and Non-Transformed Bacteria

![](https://static.igem.org/mediawiki/2019/8/83/T--Lund--Relative_ars.svg)

Figure 11: Relative concentration of arsenite in growth media at different timepoints (0, 2.5 and 5 h) with transformed and untransformed (control) strains of *E. coli*.

#### Discussion

Arsenite does not suffer from sorption to glass surfaces as it forms oxy ions which are partly dissociated leading to negatively charged ions [5]. Therefore, this decrease cannot be accounted for by the sticking of ions to the glass surface. It might be the case that the engineered bacteria, as well as the control, have a capacity to uptake arsenite. According to Chen et al., when the cells are depleted of endogenous energy reserves, arsenite enters whether or not the cells carry a resistance plasmid [7]. The ars operon carried on the Escherichia coli R-factor R773 encodes the transport system that extrudes arsenite, and the lowering of the intracellular concentration of toxic oxyanion produces resistance. However, plasmid lacking strains of *E. coli* have also been shown to be intrinsically resistant to arsenite due to the presence of chromosomal ars operon. It has also been found that the R773 *arsR* protein is 75 % identical with the chromosomal product in *E. coli* strains [6]. The wild type *E. coli* strains are resistant to arsenite up to a concentration of 1 mM. This might explain the similarity in growth and arsenite uptake between the engineered EcN and BL21(DE3) strains, and the wild type strains. However, if the measured arsenite concentration at 0 hour is compared with the concentration at 2.5 hours, the engineered BL21(DE3) shows a 21 % accumulation of arsenite, compared with only 8 % arsenite accumulation in the control. Similarly, for EcN, the comparison of measured arsenite concentration at 0 hours with the concentration at 2.5 hours, shows that the engineered EcN accumulated 22 % arsenite, whereas the control accumulated 13 % arsenite. This can be considered as a proof of concept; showing that the engineered EcN and BL21(DE3) strains express *arsR* and have an improved ability to uptake arsenite. The slight increase in the arsenite concentration at 5 hours might suggest that some cells died and released the accumulated arsenite. The growth curves in figure 10 and figure 11 imply that the wild type, as well as engineered strains were able to survive in the presence of arsenite and at pH 5. However, EcN had a very low growth rate at pH 3, suggesting that the cumulative effect of low pH and the presence of toxic arsenite might be inhibitory for the cells.

### Investigation of Composite Parts for Lead Accumulation

pbrD-T7 composite part consists of a T7 promoter [(BBa\_I719005)](http://parts.igem.org/Part%3ABBa_I719005), two Anderson RBS [(Part:BBa\_J61109)](http://parts.igem.org/Part%3ABBa_J61109), separating two coding regions of lead binding protein [(BBa\_K3282002)](http://parts.igem.org/Part%3ABBa_K3282002) and lead transport protein [(BBa\_K3282001)](http://parts.igem.org/Part%3ABBa_K3282001), followed by a TE terminator [(Part:BBa\_B0012)](http://parts.igem.org/Part%3ABBa_B0012), whereas the [pbrD-Tac](http://parts.igem.org/Part%3ABBa_K3282007) (pbrD-Tac) comprises the same basic parts but with a Tac promoter ([BBa\_K3282006](http://parts.igem.org/Part%3ABBa_K3282006)).

The *pbrD* gene encodes a Pb(II)-binding protein which is essential for functional lead sequestration, whereas the expression of *pbrT* leads to uptake of lead into the cytoplasm to reduce interaction of free Pb(II) with side chains of membrane and periplasmic proteins, which would cause extensive cellular damage [8].

#### Experiment

The [pbrD-T7](http://parts.igem.org/Part%3ABBa_K3282005) part was inserted into pUC19 and successfully transformed in *E. coli* BL21(DE3), and the [pbrD-Tac](http://parts.igem.org/Part%3ABBa_K3282007) part was transformed in the probiotic strain EcN. *pbrD* and *pbrT* protein expression was demonstrated by performing SDS-PAGE. The cells were cultured in enriched media (containing peptone, yeast extract, phosphates, chlorides and sulphates) at 37 °C. They were grown overnight and were harvested in their late exponential phase to normalize the OD values to 2. After normalization, the cells were resuspended in fresh enriched medium containing 10 µM, 50 µM, and 200 µM of Pb(NO3)2. As a negative control, the experiment was also performed with the wild type *E. coli* BL21(DE3), and wild type EcN in growth medium containing 50 µM Pb(NO3)2.

Samples were collected every hour for OD measurement. For investigating the arsenic accumulation by engineered bacteria, samples were collected at 0 hours, 2.5 hours and 5 hours after the addition of Pb(NO3)2.

#### Results

The SDS-PAGE analysis result can be seen in figure 12 and figure 13. The PbrD is a 26.7 kDa and PbrT 68.3 kDa protein. Therefore, the bands were expected to be just above the 25kDa band, and just below the 70 kDa band for PbrD and PbrT respectively. The gel shows that the engineered bacteria, as well as the control, have bands with the expected molecular weight. Thus, we can not confirm that the bands corresponds to the desired proteins. It is possible that there are other proteins expressed in *E. coli* with the same molecular weight, masking the expression of *pbrD* and *pbrT*.

![](https://static.igem.org/mediawiki/2019/f/f2/T--Lund--SDSPAGE_BL21_Pb.png)

Figure 12: Samples were harvested at different time points, and the total amount of cellular proteins were analyzed by SDS-PAGE. Image with protein bands of BL21(DE3) after induction with IPTG, showing pbrD at 26.7 kDa and pbrT at 68.3 kDa. The control shown is wild type BL21(DE3) without *pbrD* and *pbrT*.

![](https://static.igem.org/mediawiki/2019/f/f1/T--Lund--SDSPAGE_EcN_Pb.png)

Figure 13: Samples were harvested at different time points, and the total amount of cellular proteins were analyzed by SDS-PAGE. Image with protein bands of BL21(DE3) after induction with IPTG, showing PbrD at 26.7 kDa and pbrT at 68.3 kDa. The control shown is wild type BL21(DE3) without *pbrD* and *pbrT*.

##### Lead Accumulation Over Time

![](https://static.igem.org/mediawiki/2019/d/de/T--Lund--lead_accumulation.svg)

Figure 14: Lead accumulation over time. The red line represents the lead concentration in a culture that contains the composite part, while the blue line represents the lead concentration in a culture with wild type *E. coli* BL21(DE3).

##### Lead Accumulation Over Time at Varying pH

![](https://static.igem.org/mediawiki/2019/f/fa/T--Lund--lead_varying_pH.svg)

Figure 15: Lead accumulation over time. The blue line represents the lead concentration in a culture maintained at pH 3 containing the composite part, while the red line represents lead concentration in a culture maintained at pH 5 containing the composite part. The yellow line represents the lead concentration in a culture with wild type EcN.

Figure 14 and figure 15 compares the lead accumulation over time, whereas figure 16 and figure 17 compares the growth rates of engineered as well as wild type strains. It can be seen in figure 14 that the initially added lead concentration to both engineered and wild type cells was 10.63 mg/L, and the concentration at 0 hours was 0.63 mg/L and 0.66 mg/L respectively. Similarly, it can be seen in figure 15 that the initially added lead concentration was 41.44 mg/L, maintained at pH 3 and the concentration at 0 hours was 6 mg/L, showcasing an 86 % decrease. The culture maintained at pH 5 with an initial lead concentration of 10.36 mg/L also showed an 89 % decrease in lead concentration at time 0 hours. In comparison, the control shows no decrease in lead concentration. The overall lead accumulation was calculated to be 41 % by the engineered EcN, and 40 % by the engineered BL21(DE3) after 2,5 hours. The relative concentration of lead in media where transformed and non transformed bacteria were present is showcased in figure 18.

##### Growth comparison between transformed and non-transformed *E. coli* BL21(DE3)

![](https://static.igem.org/mediawiki/2019/a/a2/T--Lund--pb_growth_BL21.svg)

Figure 16: Comparison of the growth rate of engineered BL21(DE3) with wild type BL21(DE3) in the presence of lead nitrate over 6 hours.

##### Growth comparison between transformed and non-transformed EcN

![](https://static.igem.org/mediawiki/2019/d/da/T--Lund--pb_growth_EcN.svg)

Figure 17: Comparison of the growth rate of engineered EcN with wild type EcN in the presence of lead nitrate over 6 hours.

##### Relative Concentration of Lead in Media Containing Transformed and Non-Transformed Bacteria

![](https://static.igem.org/mediawiki/2019/2/20/T--Lund--Relative_PB.svg)

Figure 18: Relative concentration of lead in growth media at different time points (0, 2.5 and 5 h) with transformed and untransformed (control) strains of *E. coli*.

#### Discussion

The rapid decrease in the concentration as seen in figure 14 and 15 is similar to the decrease in arsenite concentration as explained in the previous discussion. A percentage of this rapid decrease can be attributed to the use of borosilicate Erlenmeyer flasks, since it was found that borosilicate glass can adsorb lead ions [9]. However, the study also mentions that adsorption of lead by borosilicate glass is time-dependent and adsorption was found to be only 17.2% over a period of 20 hours. This concludes that not all the lead adsorption was by borosilicate glass but also by the bacterial strains. The slight increase in lead concentration at hour 5 in figure 14 can be explained by the death of cells and release of the accumulated lead. Figure 16, 17 and 18 indicate that the engineered cells are capable of accumulating lead and can survive under conditions simulated.

## In Conclusion

Our results indicate an increase in the bacteria capability to bioaccumulate the toxic metals when the composite parts were introduced. However, due to lacking data points we cannot establish statistical significance. Our composite parts and our concept of using microbes to accumulate toxins in the human body deserves further investigation.

## Future Plans

We have failed on the following points:

* To see evidence of protein expression using SDS-page.
* To sequence the parts [BBa\_K3282005](http://parts.igem.org/Part%3ABBa_K3282005) (pbrD-T7) and [BBa\_K3282007](http://parts.igem.org/Part%3ABBa_K3282007) (pbrD-Tac).

More testing needs to be conducted in order to establish a significant result regarding the effeciveness of our composite parts. If significance is found, the bacteria should be tested *in vivo*, possibly in worms as we first planned. From thereon we can use the experimental data to validate our model further, in order to safely predict the outcome if applied in humans. A viable solution and risk-assessement for the leakage of GMMs into the environment needs to be found before further trials regaring animal and human trials may begin.

Our concenpt of using microbes to remove toxins from the human body is worthy of further exploration. It may be a viable solution in removing pesticides and carcinogenic chemicals.

#### References:

* [1] Splichalova, A., Splichal, I., Sonnenborn, U., & Rada, V. (2014). A modified MacConkey agar for selective enumeration of necrotoxigenic E. coli O55 and probiotic E. coli Nissle 1917. *J Microbiol Methods, 104*, 82-86. doi:10.1016/j.mimet.2014.06.017
* [2] Barth, S., Duncker, S., Hempe, J., Breves, G., Baljer, G., & Bauerfeind, R. (2009). Escherichia coli Nissle 1917 for probiotic use in piglets: evidence for intestinal colonization. *Journal of Applied Microbiology, 107*(5), 1697-1710. doi:10.1111/j.1365-2672.2009.04361.
* [3] Blum-Oehler, G., Oswald, S., Eiteljörge, K., Sonnenborn, U., Schulze, J., Kruis, W., & Hacker, J. (2003). Development of strain-specific PCR reactions for the detection of the probiotic Escherichia coli strain Nissle 1917 in fecal samples. *Res Microbiol, 154*(1), 59-66. doi:[https://doi.org/10.1016/S0923-2508(02)00007-4](https://doi.org/10.1016/S0923-2508%2802%2900007-4)
* [4] Kostal, J., Yang, R., Wu, C. H., Mulchandani, A., & Chen, W. (2004). Enhanced arsenic accumulation in engineered bacterial cells expressing ArsR. *Applied and environmental microbiology*, *70*(8), 4582–4587. doi:10.1128/AEM.70.8.4582-4587.2004
* [5] Masee, R., Maessen, F.J.M.J. (1981). Losses of silver, arsenic, cadmium, selenium, and zinc traces from distilled water and artificial sea-water by sorption on various container surface. Analytica Chimica Acta, 127 , pp. 206-210
* [6] Carlin, A., Shi, W., Dey, S., & Rosen, B. P. (1995). The ars operon of Escherichia coli confers arsenical and antimonial resistance. *Journal of bacteriology*, *177*(4), 981–986. doi:10.1128/jb.177.4.981-986.1995.
* [7] Chen, C., Misra, T., Silver, S., Rosen, B. (1986). Nucleotide sequence of the structural genes for an anion pump: the plasmid-encoded arseniteal resistance operon. . J. Biol. Chem., 261, pp. 15030-15038.
* [8] Borremans, B., Hobman, J. L., Provoost, A., Brown, N. L., & van Der Lelie, D. (2001). Cloning and functional analysis of the pbr lead resistance determinant of Ralstonia metallidurans CH34. *Journal of bacteriology*, *183*(19), 5651–5658. doi:10.1128/JB.183.19.5651-5658.2001
* [9] Kim N. D., & Hill, S. J. (1993). Sorption of lead and thallium on borosilicate glass and polypropylene: Implications for analytical chemistry and soil science. *Environ Technol, 14*(11), 1015-1026. doi:10.1080/09593339309385378

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