iGEM 2022 — Team wrightstate

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#### **Meeting Dates**

* 05/17/22 General "what is iGEM" talk and "what is synthetic biology"
* 05/24/22 Continued "What is synthetic Biology" discussion
* 05/31/22 Talked about plasmids and drew images
* 06/7/22 Began brainstorming
* 6/14/22 Brainstorming continued
* 6/21/22 Literature Review
* 6/28/22 Literature Review
* 7/5/22 Landed on the idea of a biosensor and moved onto investigating the different contaminents
* 7/12/22 Looked into common water contaminents
* 7/19/22 Literature review
* 7/26/22 Assembled sequences with Dr. Amy Breedon
* 8/2/22 Collected Data
* 8/9/22 Collected Data
* 8/16/22 Planning outreach with Boonshoft and COSI
* 8/25/22 Laboratory troubleshooting
* 9/1/22 Strategized final presentation
* 9/8/22 Talked about story of a presentation
* 9/15/22 Trained team on Boonshoft protocols
* 9/22/22 Performed Boonshoft Outreach
* 9/29/22 Trained team on COSI protocols
* 10/6/22 Planning COSI outreach
* 10/13/22 Presentation planning
* 10/20/22 Presentation planning
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#### **Design**

In *E. coli*, there naturally exists a pathway that the organism uses to fine-tune the concentration of manganese within the cell that revolves around the Manganese Transport Regulator (MntR) protein. When designing our gene block, we used the mntP (exporter) promoter and riboswitch in this pathway that respond to manganese. As a response to the manganese, we made our gene block produce superfolder GFP (sfGFP) as a simple reporter. Finally, we put a BBa\_B0015 terminator at the end of our gene block. This is a “commonly used” and “reliable” double terminator (B0010-B0012) from the iGEM registry.

We also built another construct to control expression of MntR. The lac operon in our backbone is inducible by IPTG, allowing precise control over how much mntR is generated by the cell. This could help us control the sensitivity of our sensor. The MntR produced by this construct will contain a 6xHIS tag to aid in back-end testing, such as in immunoblotting.

#### **Build**

We had a pTrc backbone with an insert transformed into bacteria from last year’s project. This plasmid contained cut sites for NcoI and HindIII surrounding the gene block. These enzymes were used to excise the old gene block and leave us with linearized pTrc backbone. Using HiFi cloning and the 20bp regions of homology between our gene block and our backbone, we cloned the 6xHIS-mntR overexpression gene block into the plasmid and transformed the DNA into MG1655 WT *E. coli*.

In addition, last year, we received dried pSB3K3-RFP plasmid in a distribution from iGEM. This dried plasmid was resuspended in water and transformed into NEB5α cells. EcoRI and SpeI were used to cut pSB3K3 and remove the RFP gene. We used HiFi Assembly to insert our sensor gene block (pmntP-rs-sfGFP), received from IDT, into pSB3K3. We used a New England Biolabs kit to do the assembly as well as the subsequent transformation into provided MG1655 WT *E. coli* cells.

#### **Test**

Our initial manganese screening involved a dilution series of manganese (II) chloride0.001mM – 110mM. Borrowing a protocol from the Air Force Research Laboratory, we grew cells at 37°C at 250rpm for 2 hours toan A 600 of 0.5. Manganese chloride solution was added to the cultures in a log dose series of concentrations from 0.001mM – 110mM. A treatment similar to that of the MG1655 cells was performed on a positive GFP control (pET29b-eGFP) with 1mM IPTG to induce transcription. Cultures of 1.5mL were treated with 150uL of 1M IPTG and left in incubation for 3 hours. While the fluorescence was not visible by pelleting the bacteria and examining on a UV table, we were able to measure fluorescence using our BioTek Synergy H1 plate reader. The dose response curve showed that our pSB3K3-pmntP-rs-sfGFP sensor in wild-type MG1655 E.coli produced sfGFP in response to manganese chloride in the low mM range (1-10mM). The higher tested doses (not shown) inhibited the growth of E.coli. The plasmid was also transformed into MG1655 cells with MntR deleted, a strain in the E.coli Keio collection obtained from Horizon Discovery (part of Perkin Elmer) and generated by Baba et al 2006. When treated with manganese, these cells did not produce fluorescence seen in wild type cells, indicating that the sensor was functioning in a manner dependent on the MntR pathway, and thus specific to manganese levels.

#### **APA Cite**

Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., Datsenko, K. A., Tomita, M., Wanner, B. L., & Mori, H. (2006). Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Molecular systems biology, 2, 2006.0008. https://doi.org/10.1038/msb4100050

 ![](https://static.igem.wiki/teams/4217/wiki/engineering/engineering-success-updated-figure.jpg)

#### **Learn**

Our sensor has an active range between 0.1-10mM. In subsequent tests, we will test additional concentrations in this range. By doing this, we will both flesh out the dose-response curve as well as find what concentration of manganese becomes lethal to the cells. In addition, we had large amounts of background fluorescence in our readings. LB media is made with yeast extract, so we suspect it inherently contains manganese. An alternative media that we could try is M9, a minimal salts media with no manganese. Finally, our sensor is not sensitive enough to detect low, dangerous levels of manganese. The upper limit for safe manganese levels is 0.5ppm (O'Neal), which is equal to 0.009 mM. Our sensor only goes down to 0.1mM. We will try to use pTrc-mntR to control the amount of MntR in the cell and improve the sensitivity of the sensor.

#### **Sources:**

BBa\_B0015 Link: https://parts.igem.org/Part:BBa\_B0015

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4545267/

O'Neal SL, Zheng W. Manganese Toxicity Upon Overexposure: a Decade in Review. Curr Environ Health Rep. 2015 Sep;2(3):315-28. doi: 10.1007/s40572-015-0056-x. PMID: 26231508; PMCID: PMC4545267.

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## **Experiments**

### **Standard protocols**

---

The following kits were used according to the manufacturer’s recommendations. Variations from the recommended protocol are noted as appropriate.

* NEB Plasmid Monarch Miniprep (# T1010S)
* Monarch® DNA Gel Extraction Kit Protocol (NEB #T1020S)
* Omega Biotek E.Z.N.A Plasmid DNA Maxi kit (#D6922-02)li>
  *Deviation from protocol:*

  Two additional spins were included prior to elution to remove residual ethanol containing wash buffer. Elutions were made in 1 mL water heated to 80°C to improve yield.

  * NEBuilder® HiFi DNA Assembly Cloning Kit (# E5520S)
  * Q5® High-Fidelity 2X Master Mix (# M0492S)

### **Reagent Stock Solutions**

---

#### **Antibiotic Stocks:**

* 100 mg/mL Ampicillin (1000x) in water, stored at -20°C
* 50 mg/mL Kanamycin (1000x) in water, stored at -20°C

#### **Chemical and Reagent Stocks:**

- **1.5M Manganese Chloride:** 14.69g manganese (II) chloride tetrahydrate (MW 197.90g/L) added to a final volume of 100ml and sterile filtered through a 0.22 µm syringe filter. Stored at room temperature.
- **1M IPTG:** 2.38 g of IPTG added to 8 mL of molecular biology grade H2O to dissolve, and then brought to a final volume of 10 mL with molecular biology grade H2O. Filter sterilized with a 0.22μ syringe filter. Stored in 1mL aliquots at -20 °C.
- M9 medium (VWR, #10128-268)
- Anti-GFP antibody (Santa Cruz, sc-9996)
- Anti-HIS antibody (Cell Signaling Technologies, # 2365S)
- Quick-Load® Purple 1 kb Plus DNA Ladder (#N0550S)

### **HiFi cloning of pSB3K3-pmntP-rs-sfGFP geneblock into pSB3K3**

---

The pSB3K3 backbone and pmnTP-rs-sfGFP geneblock were assembled and transformed into NEB5α using the NEB HiFi Assembly Cloning Kit (catalog # E5520S) according to manufacturer’s instructions with an approximate vector:insert ratio of 1:2. A 20ul reaction was set up as indicated below and incubated in a thermocycler at 50°C for 15 minutes

|  | Volume | Stock   Conc. | Quantity   in Reaction |
| --- | --- | --- | --- |
| Insert | 7.8 µL | 10 ng/µL | 78 ng |
| Vector | 1.6 µL | 38.6 ng/µL | 61.8 ng |
| Master Mix | 10 µL | 2x | 1x |
| Water | 0.6 µL | - | - |
| Total | 20 uL | - | 20 uL |

After assembly, the reaction was transformed into NEB 5-alpha cells as recommended. Resulting colonies were screened by restriction digest. To do so, miniprep DNA was prepared from 3ml overnight cultures by NEB Monarch miniprep kit (catalog # T1010S) according to manufacturer’s instructions. Restriction digests were performed as indicated in the **“Restriction Digest”** methods.

### **Restriction digest of pSB3K3-RFP and pSB3K3-pmntP-rs-sfGFP using EcoRI and SpeI.**

---

Plasmid DNA was digested with EcoRI, SpeI-HF or both in order to (1) obtain the pSB3K3 plasmid backbone for HiFi cloning, and (2) to confirm insertion of the pmntP-rs-sfGFP sensor geneblock into pSB3K3. Digests were set up using 400ng - 3 µg of plasmid DNA and digested for 4hr at 37C in a thermocycler followed by heat-inactivation of restriction enzymes at 80°C for 20 min. The table below shows a typical digest setup using ~400ng plasmid DNA. (The amount of enzyme was adjusted based on the amount of template used.) Subsequently, 25 μl of each reaction were run on a 0.8% agarose gel and imaged on a Fuji LAS 4000.

| Material | EcoR1 | SpeI-HF | Double | Concentration /   Activity |
| --- | --- | --- | --- | --- |
| Plasmid | 9.3uL | 9.3uL | 9.3uL | 41.6ng/uL |
| 10x Cutsmart | 5uL | 5uL | 5uL | 10x |
| EcoRI | 0.25uL | 0uL | 0.25uL | 20U/uL |
| SpeI-HF | 0uL | 0.25uL | 0.25uL | 20U/uL |
| Water | 35.45uL | 35.45uL | 35.2uL | - |
| Total | 50uL | 50uL | 50uL | - |

### **Restriction digest of pTrc source plasmid (pTrc\_VvTs\_trGppsGB from WSU iGEM 2021) and pTrc-6X-HIS-mntR using EcoRI and SpeI.**

Plasmid DNA was digested with EcoRI, SpeI-HF or both in order to (1) obtain the pSB3K3 plasmid backbone for HiFi cloning, and (2) to confirm insertion of the pmntP-rs-sfGFP sensor geneblock into pSB3K3. Digests were set up using 400ng - 3µg of plasmid DNA and digested for 4hr at 37C in a thermocycler followed by heat-inactivation of restriction enzymes at 80°C for 20 min. The table below shows a typical digest setup using ~400ng plasmid DNA. (The amount of enzyme was adjusted based on the amount of template used.) Subsequently, 25ul of each reaction were run on a 0.8% agarose gel and imaged on a Fuji LAS 4000.

| Material | NcoI | HindIII-HF | Double | Concentration / Activity |
| --- | --- | --- | --- | --- |
| Plasmid | 2.8uL | 2.8uL | 2.8uL | 351.1ng/uL |
| 10x   Cutsmart | 5uL | 5uL | 5uL | 10x |
| NcoI | 0.5uL | 0uL | 0.5uL | 20U/uL |
| HindIII-HF | 0uL | 0.5uL | 0.5uL | 20U/uL |
| Water | 41.7uL | 41.7uL | 41.2uL | - |
| Total | 50uL | 50uL | 50uL | - |

### **PCR of pTRC-6X-HIS-mntR insert to verify presence of insert**

---

PCR was used in conjunction with restriction digest and sequencing as a confirmation of 6X-HIS-mntR insert in the pTrc plasmid backbone. The reaction was set up using the following primers:

**Forward:** 5’-cggttctggcaaatattctg-3’ (45% GC, Tm 51 °C)

**Reverse: 5’-ctgatttaatctgtatcaggctg-3’ (39% GC, Tm 51°C)**

PCR was set up in thin-walled PCR tubes using Q5 High-Fidelity 2X Master Mix (catalog # M0492S) according to manufacturer’s recommendations as follows:

| Material | No DNA Neg control | Plasmid Template | Stock Concentration |
| --- | --- | --- | --- |
| Template | 0uL | 0.5uL | 79.2ng/uL |
| Forward   Primer | 1.25uL | 1.25uL | 10uM |
| Reverse   Primer | 1.25uL | 1.25uL | 10uM |
| Q5   High Fidelity Master Mix | 12.5uL | 12.5uL | 2X |
| Water | 10.0uL | 9.5uL | - |
| Total | 25uL | 25uL | - |

98°C 30sec

98°C 10sec <--|

54°C 15sec 35 cycles

72°C 30sec <--|

72°C 2min

4°C hold

The PCR products were run on a 0.8% agarose gel for 1 hour. 5ul of 1kb DNA ladder was loaded, and the entire PCR sample for each reaction.

### **Preparation of chemically competent MG1655 ΔmntR E.coli**

---

The following solutions were made:

* 100 mL of 0.1M CaCl2
* 60 mL of 0.1M CaCl2 + 15% glycerol

Protocol:

* 1 mL of an overnight culture of ΔmntR E. coli in LB medium (no antibiotic) was diluted in 99 mL of LB and incubated at 37°C, 200rpm until the OD600 reached 0.4.
* CaCl2 solutions and pre-labeled ΔmntR tubes were placed on ice before starting.
* The culture was separated evenly into 3 Oakridge tubes and centrifuged at 4°C, 4000rpm for 10 minutes.
* The supernatants were aspirated, and pellets were resuspended in 0.1 M CaCl2 and incubated on ice for 30 min.
* The samples were centrifuged for 10 min at 4°C, 4000rpm.
* Supernatant was dumped into bleach containers.
* Pellets were resuspended in 1 mL of 0.1M CaCl2 + 15% glycerol and combined into one tube along with another 2mL of CaCl2 + glycerol.
* One at a time, pre-chilled, pre-labeled 1.7 mL tubes were filled with 25uL of bacteria, snap frozen in liquid nitrogen, and stored on dry ice.
* 61 tubes of competent cells were stored at -80°C.
* Competency of cells was confirmed by transformation of pUC19.

### **Functional Testing of sensor pSB3K3-GB**

The general outline of the sensor assay is shown below. The basic assay was initiated from a glycerol stock of the plasmid(s) of interest in wild-type MG1655 *E. coli*. A 5 mL culture was set up in LB with 50 µg/mL Kanamycin and grown in a shaker at 250 RPM overnight at 37°C. This culture was back-diluted into a larger working culture (20-100 mL) of media with antibiotic and grown to an OD600 of 0.5. Typically, cultures were aliquoted into 1-5 mL volumes in culture tubes for treatment with manganese chloride. After incubation with MnCl2M/sub>, 100 µl volumes were added to shielded microwell plates for A600 and Fuor485/515 readings.

![](https://static.igem.wiki/teams/4217/wiki/experiments/experiments-graphic-updated.png)
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## **Goal: Build a biosensor construct that will respond to manganese in solution**

**Summary of Overall Approach:** The following summary of experimental results highlights the experiments performed to develop and test a functional manganese sensor. Our approach involved two gene block constructs derived from manganese homeostatic signaling systems native to E. coli. The first included the pmntP promoter that responds to MntR after it has bound to Mn(II) and a riboswitch that forms a hairpin and blocks translation of mRNA unless in the presence of Mn(II). We chose to clone the sensor geneblock (pmntP-rs-sfGFP) into the pSB3K3 plasmid backbone which contains a KAN resistance gene and the required elements for expression in MG1655 *E. coli*.

In addition, we constructed a plasmid for the IPTG inducible expression of 6xHIS-tagged MntR. This approach provides control over the levels of the manganese-responsive transcription factor MntR and a 6xHIS tag to facilitate monitoring of its expression level. This geneblock was inserted into a separate plasmid backbone containing the pTrc promoter with a lac operator that allows us to treat with IPTG and control the amount of MntR in the cells. This plasmid contains an AMP resistance gene.

## **“pSB3K3-pmntP-rs-sfGFP” Sensor Design**

A 1796-nucleotide geneblock was ordered from Addgene which incorporates 5’ and 3’ homology to the pSB3K3 vector to facilitate HiFi cloning **(Fig. 1)**. The geneblock contains the full *E. coli* mntP promoter (BBa\_K4217000). The full promoter has been shown to be more effective than an alternate truncated form which was previously used by the Calgary 2020 iGEM team (BBa\_K902073). The manganese-responsive riboswitch (BBa\_K902074), sfGFP (BBa\_I746916), T1 and T7 terminator (BBa\_K902073) were placed downstream of the mntP promoter.

![](https://static.igem.wiki/teams/4217/wiki/results-images/construct-1-psb3k3-pmntp-rs-sfgfp.png)

**Fig. 1. Description of the pSB3K3-pmntP-rs-sfGFP sensor plasmid.**

## **Experimental Summary of Steps Taken in the Cloning of sensor pSB3K3-pmntP-rs-sfGFP:**

1. **Cut mRFP insert out of pSB3K3-mRFP backbone using EcoRI and SpeI.**

pSB3K3-RFP plasmid DNA was purified from an overnight 250ml culture of pSB3K3-RFP in LB media with 50µg/ml Kanamycin using the Omega E.Z.N.A. Plasmid DNA Maxi Kit (catalog #D6922-02) according to manufacturer’s instructions. A final concentration of 87.1ng/µL (1.82 A260/280, 1.79 A260/230) was obtained and subsequently used for restriction digest as shown in the following table:

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Material | EcoRI | SpeI | Double Digest | Concentration   / Activity |
| pSB3K3-RFP | 11.5 µL | 11.5 µL | 11.5 µL | 41.6ng/µL |
| 10X CutSmart | 5 µL | 5 µL | 5 µL | 10x |
| EcoRI | 0.5 µL | 0 µL | 0.5 µL | 20U/µL |
| SpeI-HF | 0 µL | 0.5 µL | 0.5 µL | 20U/µL |
| Water | 33 µL | 33 µL | 32.5 µL | - |
| Total | 50 µL | 50 µL | 50 µL | - |

The resulting digest was run on a 0.8% gel, and the backbone was gel purified using the Monarch DNA Gel Extraction Kit (#T1020S).

2. **HiFi cloning of pmntP-rs-sfGFP geneblock into linearized pSB3K3 backbone**

The pSB3K3 backbone and pmntP-rs-sfGFP geneblock were assembled and transformed into NEB5α using the NEB HiFi Assembly Cloning Kit (catalog # E5520S) according to manufacturer’s instructions.

3. **Confirmation of target plasmid using EcoRI and SpeI.**

Approximately 400ng of plasmid DNA was digested with EcoRI, SpeI-HF or both in order to confirm insertion of the sensor geneblock into pSB3K3. The digest was set up as follows and incubated for 4hr at 37°C in a thermocycler. Subsequently, 25 µL of each reaction was run on a 0.8% agarose gel and imaged on a Fuji LAS 4000.

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Material | EcoR1 | SpeI-HF | Double   Digest | Concentration / Activity |
| Plasmid | 9.3µL | 9.3µL | 9.3µL | 41.6ng/µL |
| 10x Cutsmart | 5µL | 5µL | 5µL | 10x |
| EcoRI | 0.25µL | 0µL | 0.25µL | 20U/µL |
| SpeI-HF | 0µL | 0.25µL | 0.25µL | 20U/µL |
| Water | 35.45µL | 35.45µL | 35.2µL | - |
| Total | 50µL | 50µL | 50µL | - |

![](https://static.igem.wiki/teams/4217/wiki/results/nrr-2.jpg)

**Fig. 2. Restriction digest confirmation of the pSB3K3-pmntP-rs-sfGFP plasmid.** 5µL of Purple 1Kb Plus DNA ladder (#N0550S) and 25µL of each sample was run on a 0.8% agarose gel and imaged on a Fuji LAS 4000.

**Conclusions:**

* The single (EcoRI and SpeI) digests confirm the target plasmid size of 4.5kb, and the double digest showing bands at 2.6kb and 1.8kb serve as confirmation that the plasmid contains both the pmntP-rs-sfGFP insert and pSB3K3 backbone, as desired.
* Additionally, the pSB3K3-pmntP-rs-sfGFP plasmid sequence was confirmed by Sanger Sequencing by GeneWiz.

## **Testing of sensor pSB3K3-pmntP-rs-sfGFP**

1. **Determine if sfGFP is indused in response to MnCl2**

The initial test of the pSB3K3-pmntP-rs-sfGFP sensor was a confirmation that sfGFP protein was produced in response to MnCl2 treatment. A 1.5M stock solution of Manganese (II) chloride tetrahydrate was diluted roughly into a 10-fold dilution series for use in the assay.

![](https://static.igem.wiki/teams/4217/wiki/results/results-4.jpg)

Overnight cultures were set up of:

1. MG1655 *E. coli* expressing the pmntP-rs-sfGFP sensor
2. A negative control culture of MG1655 ΔmntR (mutant *E. coli* lacking the MntR transcription factor required for manganese homeostasis) expressing the pmntP-rs-sfGFP sensor]
3. An IPTG-inducible positive control (pET29b-eGFP) known to express eGFP at high levels

## **“pTrc-6xHIS-mntR” Sensor Design**

Based on the observation that the pSB3K3-pmntP-rs-sfGFP sensor doesn’t work in ΔmntR mutant *E. coli* and is thus dependent on mntR, it was hypothesized that an inducible mntR plasmid would allow us to optimize sensor performance. A 535 nucleotide geneblock was ordered from Addgene which incorporates 5’ and 3’ homology to the pTrc vector to facilitate HiFi cloning **(Fig.9)**. The geneblock contains the N-terminal 6xHIS tag and the coding sequence for mntR (source). The full promoter has been shown to be more effective than an alternate truncated form which was previously used by the Calgary 2020 iGEM team (BBa\_K902073). The manganese-responsive riboswitch (BBa\_K902074), sfGFP (BBa\_I746916), T1 and T7 terminators (BBa\_B0015) were placed downstream of the mntP promoter.

 ![](https://static.igem.wiki/teams/4217/wiki/results/nrr-9.png)

**Fig. 9. Description of the pTrc-6xHIS-mntR plasmid.**

## **Experimental Summary of Steps Taken in the Cloning of pTrc-6xHIS-mntR:**

The pTrc-VvTs-trGPPS plasmid produced by the 2021 Wright State iGEM team was used as the source of the plasmid backbone used to make the pTrc-6xHIS-mntR plasmid. Plasmid DNA was purified from an overnight 250 mL culture of pTrc-VvTs-trGPPS in LB media with 100µg/ml Ampicillin using the Omega E.Z.N.A. Plasmid DNA Maxi Kit (catalog #D6922-02) according to manufacturer’s instructions. A final concentration of 351ng/µL (1.85 A260/280, 2.2 A260/230) was obtained and subsequently used for restriction digest as shown in the following table:

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Material | NcoI | HindIII | Double Digest | Concentration / Activity |
| DNA | 2.8 µL | 2.8 µL | 2.8 µL | 351.1ng/µL |
| CutSmart | 5 µL | 5 µL | 5 µL | 10x |
| NcoI-HF | 0.5 µL | 0 µL | 0.5 µL | 20U/µL |
| HindIII-HF | 0 µL | 0.5 µL | 0.5 µL | 20U/µL |
| Water | 41.7 µL | 41.7 µL | 41.2 µL | - |
| Total | 50 µL | 50 µL | 50 µL | - |

The resulting digest was run on a 0.8% gel and the backbone was gel purified using the Monarch DNA Gel Extraction Kit (#T1020S).

1. **HiFi cloning of pmntP-rs-sfGFP geneblock into linearized pSB3K3 backbone**

The pTrc backbone prepared above and the 6xHIS-mntR geneblock were assembled and transformed into NEB5α using the NEB HiFi Assembly Cloning Kit (catalog # E5520S) according to manufacturer’s instructions.

2. **Confirmation of target plasmid using Nco1 and HindIII.**

Approximately 500ng of plasmid DNA was digested with NcoI-HF and HindIII-HF or both in order to confirm insertion of 6xHIS-mntR into the pTrc backbone. The digest was set up as follows and incubated for 4hr at 37°C in a thermocycler. Subsequently, 25 µL of each reaction was run on a 0.8% agarose gel and imaged on a Fuji LAS 4000.

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Material | NcoI | HindIII | Double Digest | Concentration / Activity |
| DNA | 6.3 µL | 6.3 µL | 6.3 µL | 79.2/µL |
| CutSmart | 5 µL | 5 µL | 5 µL | 10x |
| NcoI-HF | 0.25 µL | 0 µL | 0.25 µL | 20U/µL |
| HindIII-HF | 0 µL | 0.25 µL | 0.25 µL | 20U/µL |
| Water | 38.5 µL | 38.5 µL | 38.2 µL | - |
| Total | 50 µL | 50 µL | 50 µL | - |

![](https://static.igem.wiki/teams/4217/wiki/results/nrr-10.jpg)

**Fig. 10. Restriction digest confirmation of the pTrc-6xHIS-mntR plasmid.** 5 µL of Purple 1Kb Plus DNA ladder (#N0550S) and 25 µL of each sample was run on a 0.8% agarose gel and imaged on a Fuji LAS 4000.

**Conclusions:**

* The single (NcoI and HindIII) digests confirm the target plasmid size of 4.6kb, and the double digest showing bands at 4.4kb and 230bp serve as confirmation that the plasmid contains both the 6xHIS-mntR insert and pTrc backbone, as desired.
* Additionally, the pTrc-mntR plasmid sequence was confirmed by Sanger Sequencing by GeneWiz.

## **Testing of sensor pTRC-6xHIS-mntR induction**

1. **Determine if mntR is induced in response to IPTG**

To confirm that IPTG induces the expression of mntR from the pTrc-6xHIS-mntR plasmid, several different concentrations of IPTG were tested and the induction of mntR was monitored by Coomsasie and immunoblot. Our data showed that 6xHIS mntR protein was expressed in response to 0.1 mM, 1 mM and 10 mM IPTG by 2hr and continuing through an overnight incubation (see Contribution).

2. **Determine the effect of mntR expression on the MnCl2-induced expression of sfGFP from the pSB3K3-pmntP-rs-sfGFP sensor.**

To determine the effect of mntR expression on the performance of the pSB3K3-pmntP-rs-sfGFP sensor, two colonies of double transformants (i.e. carrying both pTrc-mntR and pSB3K3-pmntP-rs-sfGFP) were subjected to the MnCl2 treatment assay **(Fig.11)**. Coomassie and immunoblot of samples from the first colony were performed as confirmation that MntR was induced in these samples **(Fig.12)**.

![](https://static.igem.wiki/teams/4217/wiki/results/nrr-11-a.jpg)

**Fig. 11: Time-course of pSB3K3-pmntP-rs-sfGFP induction in uninduced and IPTG-induced cultures co-expressing the pTrc-6xHIS-mntR plasmid.** Overnight cultures of MG1655 WT *E. coli* expressing the dual plasmid system (i.e. both the pSB3K3-pmntP-rs-sfGFP sensor and pTrc-6xHIS-mntR) were diluted 1:20 in LB-Kan+Amp and grown at 37°C 250RPM to an OD600 of 0.5. Cultures were divided into 25 mL batches and treated with either vehicle or 1mM IPTG for 2hrs at 34°C. Cultures were then aliquoted into 3 mL volumes for treatment with vehicle or 2.5 mM MnCl2. After 2hr and 4hr of treatment, A600 and Fluor485/515 readings were collected and the fold-changes relative to untreated control were calculated.

![](https://static.igem.wiki/teams/4217/wiki/results/nrr-12-a-1.jpg)

**Fig. 12. Immunoblot (top) and Coomassie (bottom) of cell lysates from MG1655 ΔmntR *E. coli* expressing the pTrc-6xHIS-mntR plasmid.** Overnight cultures of MG1655 ΔmntR *E. coli* transformed with the pTrc-6xHIS-mntR plasmid were grown in LB medium with 50 µg/ml Kanamycin, diluted 1:20 and grown to an OD600 of 0.5. The cultures were split and treated with 1mM IPTG or vehicle for 2hrs at 34°C 250RPM followed by 2 additional hours with vehicle or 2.5mM MgCl2 (as indicated). 5 µL of Low MW protein ladder (Thermo Fisher #26616) was run in the first lane. A positive control of pet29b-6xHIS-GFP was run as a positive control for IPTG induction. 0.1 OD600 equivalent of whole cell lysates were run in each lane. Proteins were separated on a 15% SDS-PAGE gel run at 135V and either stained with Coomassie stain or transferred to a 0.45 µm PVDF membrane with 0.35A for 60 min for immunoblot for the 6xHIS tag. Immunoblot was performed using mouse anti-HIS tag (Cell Signaling Technology) at 1:1000 in 5% milk overnight at 4°C and goat anti-mouse IgG at 1:5000 in 5% milk for 1 hour at room temperature. Blots were developed using Lightning Plus ECL bioluminescence substrate and imaged on a Fuji LAS 4000.

**Conclusions:**

* IPTG induction of pTrc-mntR inhibits the pSB3K3-pmntP-rs-sfGFP sensor. When prior reports that increased mntR levels inhibit the manganese importer (Kauer et al 2014 and Chen et al 2017), it appears likely that the levels of 6xHIS-mntR produced here are sufficient to cause a feedback inhibition of the Mn2+ importer and thus a drop in intracellular Mn2+ levels and sfGFP induction.
* As an experimental control, mntR expression was confirmed in IPTG treated samples **(Fig.12)**. Further, addition of 2.5mM MnCl2 did not interfere with the production of mntR.

### **Overall Findings:**

Co-expression of uninduced pTRC-6xHIS mntR with the pSB3K3-pmntP-rs-sfGFP sensor in MG1655 WT *E. coli* was confirmed as an effective cell-based system for the detection of manganese in water samples down to 0.01 mM (0.5ppm) MnCl2. This level of detection allows the testing of water samples for manganese contamination near the level at which it is visible (0.05ppm) or deemed unsafe (0.3ppm). Further, the sensor is effective down to the 1mM (5µg/L, 5ppm) practical quantitation limit (PQL) of routine water tests for manganese.

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