iGEM 2023 — Team wrightstate-oh

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{% block title %}Notebook{% endblock %}
{% block lead %}Timeline of the season's events{% endblock %}
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Experiment info here

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## Click on the blue dates to see day-to-day progress of team sub-groups

Regular team meetings were held every Thursday and every other Friday.

{% with
month = "April",
year = "2023",
important\_days = "6-7-10-12-13-15-19-20-24-25-26-27-28-29"
%}
{% include 'calendar.html' %}
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{% with
month = "May",
year = "2023",
important\_days = "4-5-9-11-16-18-19-22-23-24-25-26-30-31"
%}
{% include 'calendar.html' %}
{% endwith %}

{% with
month = "June",
year = "2023",
important\_days = "1-2-5-6-8-12-13-15-20-22-26-27-29"
%}
{% include 'calendar.html' %}
{% endwith %}

{% with
month = "July",
year = "2023",
important\_days = "6-7-13-18-19-20-21-24-26-27"
%}
{% include 'calendar.html' %}
{% endwith %}

{% with
month = "August",
year = "2023",
important\_days = "2-3-8-9-10-14-17-18-21-22-24"
%}
{% include 'calendar.html' %}
{% endwith %}

{% with
month = "September",
year = "2023",
important\_days = "1-2-6-7-8-11-13-15-18-19-20-22-25-26-28-29"
%}
{% include 'calendar.html' %}
{% endwith %}

{% with
month = "October",
year = "2023",
important\_days = "3-5-6-8-9-10-11-12-24-26"
%}
{% include 'calendar.html' %}
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{% with
month = "November",
year = "2023",
important\_days = "2-3-4-5"
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{% block lead %}The DBTL Cycle{% endblock %}
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## Overview

---

In *E. coli*, there is a well-characterized signaling pathway that regulates intracellular manganese levels. From this manganese homeostatic pathway, the 2022 Wright State iGEM team used the *pmntP* promoter and the yybP-ykoY riboswitch to make a manganese responsive plasmid. In our original design, we used sfGFP as our reporter protein due to its robust ability to quickly and correctly fold into a working fluorescent protein. To boost the performance of our sensor, we also included a plasmid for the IPTG-inducible expression of the manganese responsive transcription factor mntR. (Both are described in the [Results Wiki page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D).) Our original approach was to use whole cells in a culture-based approach to detect manganese in solution. The double plasmid approach, tested in MG1655 *E.coli* as *pSB3K3-pmntP-riboswitch-sfGFP and pTrc-6X-HIS-mntR*, was able to detect manganese down to 0.01mM, but required over 24 hours to complete, produced a small change in fluorescence in response to MnCl2, and required a plate reader for detection of manganese-induced changes in fluorescence.

To address the limitations from our 2022 approach, we decided to pursue the following engineering steps:

1. **DBTL Cycle 1**: Change to a cell-free system.- **DBTL cycle 2**: Test a T7 promoter-driven manganese biosensor.- **DBTL Cycle 3**: Test a manganese sensor utilizing a destabilized GFP reporter.- **DBTL Cycle 4**: Test a manganese sensor utilizing a NanoLuciferase (NanoLuc) reporter.

These changes were incorporated to:

1. Avoid limits due to manganese import and export which occur in a whole-cell approach.
2. Reduce assay time. By changing to a cell-free platform, we were able to avoid the overnight culture of *E.coli* required in our culture-based approach (2022).
3. Improve fieldability by providing robust, visible reporter output.

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## Stock Solutions

---

Antibiotic Stocks:

### 100mg/ml Ampicillin (1000x) in water, stored at -20°C

### 50mg/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 0.2µm syringe filter. Stored at room temperature.

### 1M IPTG

2.38 g of IPTG added to 8 mL of molecµLar biology grade H2O to dissolve, and then brought to a final volume of 10 mL with molecµLar biology grade H2O. Filter sterilized with a 0.22μ syringe filter. Stored in 1mL aliquots at -20 °C.

### Mouse monoclonal Anti-GFP antibody (Cell Signaling Technology, #2955S)

### Anti-HIS antibody (Cell Signaling Technologies, # 2365S)

### Quick-Load® Purple 1 kb Plus DNA Ladder (#N0550S)

Cell free lysate:

### Commercial: myTXTL Sigma 70 (Arbor Biosciences, # 507024).

### Homemade cell free lysate was obtained from Dr. Jorge Chavez Benavides and Dr. Kathryn Beabout from the Air Force Research Laboratories

## Equipment

---

### BioTek Synergy H1 Plate Reader capable of fluorescent, luminescent, and absorbance readings

### Fuji LAS 400 for chemiluminescence, ethidium bromide and white light imaging

### 3D printed Luminescence Imaging Device (LID) described in the Hardware Wiki.

## Mfr. 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 (NEB #T1020S)

### E.Z.N.A Plasmid DNA Maxi kit (Omega Biotek #D6922-02)

*Deviation from protocol:* Two additional spins were included prior to elution to remove residual ethanol containing wash buffer. Elutions were made in 1ml water heated to 80oC to improve yield.

### NEBuilder® HiFi DNA Assembly Cloning Kit (NEB # E5520S)

### Q5® High-Fidelity 2X Master Mix (NEB # M0492S)

### myTXTL Sigma 70 Master Mix Kit for Cell-free Expression (Arbor Biosciences # 507024)

### Nano-Glo Luciferase Assay (Promega # N1110)

## Restrict Digest

---

Geneblocks (pmntR-riboswitch-deGFP, pmntR-riboswitch-NanoLuc, and T7-riboswitch-sfGFP) were cloned into a linearized pSB3K3 backbone. To obtain the linearized backbone, 1mg of pSB3K3 plasmid DNA was digested with EcoRI and SpeI-HF. Single digests were used to confirm each enzyme cut the plasmid backbone as expected. Digests were set up using 1 microgram of plasmid DNA and digested for 3hr at 37°C in a thermocycler followed by heat-inactivation of restriction enzymes at 80°C for 20 min. The table below shows a typical digest. (The amount of enzyme was adjusted based on the amount of template used.) Subsequently, 50 µL of each reaction were run on a 0.8% agarose gel and imaged on a Fuji LAS 4000 and the desired linearized backbone was purified using the Monarch® DNA Gel Extraction Kit using the manufacturer's protocol.

|  | EcoR1 | Spel-HF | Double | Concentration/Activity |
| --- | --- | --- | --- | --- |
| **Plasmid** | 3.5µL | 3.5µL | 3.5µL | 287ng/µL |
| **10x Cutsmart** | 5µL | 5µL | 5µL | 10x |
| **EcoRI** | 0.5µL | 0µL | 0.5µL | 20U/µL |
| **Spel-HF** | 0µL | 0.5µL | 0.5µL | 20U/µL |
| **Water** | 41µL | 41µL | 40.5µL | - |
| **Total** | 50µL | 50µL | 50µL | - |

The above was also used to screen individual colonies from HiFi cloning to confirm insertion of the desired geneblock (pmntR-riboswitch-deGFP, pmntR-riboswitch-NanoLuc,
T7-riboswitch-sfGFP and pmntR-riboswitch-eforRed).

## HiFi Cloning

---

The linearized pSB3K3 backbone and individual geneblock (pmntR-riboswitch-deGFP, pmntR-riboswitch-NanoLuc, T7-riboswitch-sfGFP or pmntR-riboswitch-eforRed) were assembled and
transformed into NEB5α using the NEB HiFi Assembly Cloning Kit (catalog # E5520S) according to the manufacturer's instructions with an approximate insert:backbone ratio of 3:1.
A typical 20µL reaction was set up as indicated below and incubated in a thermocycler at 50°C for 15 minutes.

|  | Volume |
| --- | --- |
| Insert | 5µL |
| Vector | 0.5µL |
| 2x Master Mix | 10µL |
| Water | 4.5µL |
| Total | 20µL |

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 the manufacturer's instructions. Restriction digests were performed as indicated in the
**“Restriction Digest”** methods.

## Transformation

---

1. Chemically competent cells (MG1655 or NEB5 alpha) were thawed on ice.
2. Two microliters of the chilled assembled HiFI cloning reaction were added to the competent cells. Tubes were mixed gently by pipetting up and down or by flicking
   the tube 4–5 times. (Tubes were not vortexed.)
3. The mixture was placed on ice for 30 minutes.
4. Samples were heat shocked at 42°C for 30 seconds.
5. Tubes were placed on ice for 2 minutes.
6. 950 μl of room-temperature SOC media was added to each tube.
7. Tubes were incubated at 37°C for 60 minutes with 250 rpm rotation.
8. Agar plates were pre-warmed to 37°C.
9. 200 μl of the cells were spread on LB agar plates with the appropriate antibiotic. (LB-Kanamycin plates were used for all pSB3K3 plasmids described in this project.)
   The remainder was spun down, the media dumped off , and the pellet was resuspended in the small amount of LB remaining in the tube and spread on a second plate.
   This yielded 2 plates, one with ~20% of the bacteria and the second with ~80%, increasing the chances of obtaining isolated colonies for subsequent testing.
10. Plates were incubated overnight at 37°C and viewed the next day.

Resulting colonies were screened by restriction digest to confirm insertion of the desired geneblock (pmntR-riboswitch-deGFP, pmntR-riboswitch-NanoLuc, and T7-riboswitch-sfGFP) as indicated in the “Restriction Digest”methods. To do so, miniprep DNA was prepared from 3 mL overnight cultures by NEB Monarch miniprep kit (catalog #T1010S) according to the manufacturer's instructions. The colonies were screened from HiFi cloning as described above using double digestion with EcoRI and SpeI-HF.

## Whole-Cell Assay

---

### Whole-cell assay of manganese sensor plasmids

The standard whole-cell assay utilized by our 2022 iGEM team was performed for the pSB3K3-pmntP-riboswitch-sfGFP, pSB3K3-pmntP-riboswitch-deGFP, pSB3K3-T7-riboswitch-sfGFP, and pSB3K3-pmntP-riboswitch-NanoLuc. This assay was performed as follows:

1. A 5ml overnight culture LB with 50µg/ml Kanamycin was set up from glycerol stock of the plasmid(s) of interest in wild-type MG1655 E. coli (for all plasmids with a pmntP promoter) or BL21(DE3) E.coli (for plasmids with a T7 promoter) and grown in a shaker at 250 RPM overnight at 37oC.
2. This cµLture was back-diluted into a larger working culture (20-100ml) of media with antibiotics and grown to an OD600 of 0.5.
3. Cultures were then aliquoted into 1-5 ml volumes in culture tubes for treatment with manganese chloride.
4. At the desired timepoints, generally 2hr, 4hr, 6hr and 8hr, 100µl volumes were added to shielded 96-well microwell plates for A600 and Fluor485/515 readings. Each sample was read in triplicate using a BioTek Synergy H1 plate reader.
5. All readings were blanked against an LB control. Density-corrected fluorescence values were calculated by dividing the Fluor485/515 by A600 values, and then the fold-change in fluorescence was calcµLated versus the control sample with no manganese treatment (i.e. 0mM MnCl2 control).

### Whole-cell assay of the pSB3K3-pmntP-riboswitch-NanoLuc sensor

1. Cultures were grown as in the standard whole cell assay described above (i.e. overnight cultures diluted and grown to OD600 of 0.5) and incubated with or without manganese for the desired time (2hr - overnight).
2. At the desired endpoint, generally 24hr post addition of manganese, 10µL of each culture was transferred to a 96-well microwell plate. LB broth was again used as a blank.
3. NanoLuc substrate was prepared by diluting the furimazine substrate stock solution 1:50 in buffer provided in the Nano-Glo Luciferase Assay Kit (Promega, catalog number N1110). 10μl of NanoLuc substrate was added to each well.
4. The plate was tapped lightly to mix and read on a BioTek Synergy H1 plate reader using luminescence fiber, gain 135, signal integration of 1 sec per read, reads taken every 1 minute for a 20 minute time-course. This allowed us to assess the stability of the luminescent output and provided a readout of substrate excess or deficiency in our reactions.

## Cell-Free

---

The following protocol utilizes lysate from BL21(DE3) E. coli and buffer (“mix”) containing all of the required buffers, polymerases, nucleotides, tRNAs and energy sources. The specific buffer components are detailed in Silverman et al 2019 [1].

Note: Cell free assays performed using the Arbor Biosciences P70 kit were performed according to the manufacturer recommended protocol.

|  | Final Concentration in Reaction |
| --- | --- |
| **Template DNA\*** | 10nM |
| **Master Mix \*\*** | 1X |
| **MnCl2** | 0.01mM - 10mM MnCl2 |
| **Nuclease Free Water** | to 12.5µL |
| **Other (as needed)** | 1mM IPTG (for inducible plasmids)   3 units T7 polymerase (for T7 driven plasmids) |

\* Template DNA used in these reactions included plasmid DNA from the sensor being tested or relevant control plasmids. Sensor plasmids tested included pSB3K3-pmntP-riboswitch-sfGFP, pSB3K3-pmntP-riboswitch-deGFP, pSB3K3-T7-riboswitch-sfGFP, pSB3K3-pmntP-riboswitch-NanoLuc and pSB3K3-pmntP-riboswitch-eforRed. All plasmids were purified by Omega E.Z.N.A. maxiprep and then ethanol precipitated for use in cell-free assays to ensure template DNA was of sufficient purity and concentration.
\*\* The master mix was prepared by combining 3.65µl of buffer (“mix”) with 3.35µl cell extract per reaction.

| Assay | Plasmid | Role |
| --- | --- | --- |
| **All cell-free** | No DNA | Negative control (-C) |
| **All cell-free** | P70a(2)-deGFP (myTXTL kit +C) | Positive control (+C) (fluorescent) |
| **Test of sensor(s) with Flourescent Reporter** | pET28a(+)-6X-HIS-eGFP | IPTG-inducible +C (fluorescent) |
| pY71-T7-sfGFP | T7-driven sfGFP +C (fluorescent) |
| pJL1-sfGFP | Constitutively expressed +C (fluorescent) |
| **Tests of sensor with Luminescent Reporter** | pET28a(+)::NL | IPTG inducible +C (luminescent) |
| pET-28b (empty vector) | -C (luminescent) |

Assays performed using the homemade cell free extract were performed as follows:

1. The BioTek Synergy H1 plate reader was preheated to 29°C.
2. The cell extract, buffer (“mix”) and positive control plasmid (P70a(2)-deGFP) were thawed on ice until needed.

Note: To minimize freezing and thawing cycles, only the number of reagent tubes required to set up the desired number of cell-free reactions were thawed.

3. Cell-free Master Mix was prepared by combining 3.65µl of buffer (“mix”) with 3.35µl cell extract per reaction.
4. Directly before use, Master Mix was vortexed for 2-3 seconds and briefly spun down. If any precipitate was visible hereafter, the sample was gently resuspended by pipetting about 10 times to ensure homogeneity. CarefµL attention was paid to pipetting technique to avoid formation of bubbles and foam.
5. Each reaction was set up with a final volume of 12.5μL. On ice, components were combined in 0.6ml sterile PCR tubes according to the run needs. (Specific reaction setups varied by experiment and are detailed in the [results page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D).)
6. The assembled Cell Free reactions were vortexed for 2-3 seconds and briefly centrifuged to collect the entire volume at the bottom of the tube.
7. For sensors with a fluorescent readout, reactions were transferred 6µL per well into 96-well V-bottom microwell plates, covered with a plate sealer, and placed in the BioTek Synergy H1 plate reader for measurement. For NanoLuc luminescence measurements, the tubes containing the 12.5µL reactions were transferred to an incubator set to 29°C. Fluorescent measurements (Fluor485/515nm) were taken every 10 minutes for 24 hours.
8. For sensors with a luminescent readout, measurements were performed at the chosen endpoint by adding 0.5µl or 1.0µl of sample into a total 10µl water in appropriate wells in a V-bottom plate. 10µl of diluted NanoLuc substrate was added to each well. The plate was tapped lightly to mix and read on a BioTek Synergy H1 plate reader using luminescence fiber, gain 135, signal integration of 1 sec per read, reads taken every 1 minute for 20 minutes time-course.

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}The full story of our season's experiments{% endblock %}
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## Project Overview

---

Our 2022 iGEM team developed an E.coli culture-based biosensor for the detection of manganese contamination in drinking water. The sensor was based on the manganese homeostatic pathway present in E.coli **(Figure 1)**.

![](https://static.igem.wiki/teams/4681/wiki/results-pics/image17.png)

**Figure 1: E. coli manganese homeostatic pathway.** Manganese ions are brought into the cell through the mntH importer. Intracellular manganese ions bind to the mntR transcription factor, promoting its binding to the mntP gene promoter (PmntP) and promoting transcription of the riboswitch-mntP mRNA. Intracellular manganese binding to the riboswitch (RS) causes the riboswitch mRNA to open, allowing ribosome binding and translation of the mntP exporter protein. Figure modified from Wang et al 2015 [1].

The 2022 manganese biosensor approach utilized 2 plasmids as described below.

The main sensor plasmid, pSB3K3-pmntP-riboswitch-sfGFP, had an E.coli manganese responsive pmntP promoter and riboswitch placed upstream of a superfolder GFP (sfGFP) reporter **(Figure 2A)**. This design yielded a sensor with manganese-regulated transcription and translation of the sfGFP reporter protein.

The second plasmid, pTrc-6X-HIS-mntR **(Figure 2B)**, provided inducible expression of a 6X-HIS tagged manganese-binding transcription factor mntR. The inducible mntR plasmid was included to test whether increasing mntR levels would increase sfGFP output from the main sensor plasmid in response to manganese.
![](https://static.igem.wiki/teams/4681/wiki/results-pics/image3.png)

**Figure 2: Dual plasmid biosensor for the detection of Mn2+ contamination of drinking water.**(A) The manganese sensor plasmid incorporated the endogenous E.coli manganese responsive pmntP promoter, manganese-binding riboswitch, a ribosome binding site (RBS) and sfGFP reporter in a pSB3K3 plasmid backbone for expression in E.coli. (B) The inducible mntR plasmid was driven by the tac promoter in the pTrc plasmid and included the lac operon (lacO) for IPTG-inducible expression in E.coli.

The 2022 team carried out tests of the primary sensor plasmid and the inducible mntR plasmid in a whole-cell E.coli assay format. They found that the sensor **(Figure 2A)** fluoresced in response to 0.1mM manganese. When the inducible mntR plasmid **(Figure 2B)** was added, the team found that leaky, uninduced expression of mntR (i.e. in the absence of IPTG-induction) improved detection down to 0.01mM manganese. Surprisingly, when IPTG was added and excess mntR protein was produced, no sfGFP was produced at any manganese dose tested. The team theorized that increasing mntR levels caused a downregulation of the mntH manganese importer, and thus intracellular manganese levels.

As configured, the 2022 manganese biosensor had the following limitations:

1. Long assay time: 24 hours of culture followed by 8hr of growth with manganese.
2. Manganese uptake in live E.coli is limited by endogenous transporters.
3. Required the use of live bacteria, limiting fieldability.
4. Low fluorescent output: ~2-fold increase in fluorescence after 8hr incubation with 1mM MnCl2.

The 2023 Wright State iGEM team sought to improve the manganese biosensor using the following strategies:

1. The team sought to adapt the manganese biosensor to a cell-free platform to avoid the long assay times required by the E. coli culture-based approach. This approach was also intended to circumvent limitations in sensitivity arising from manganese transport occurring in live E.coli. This work is described in **PART 1** below.
2. To address the low fluorescent output of the original sensor in response to manganese, the team explored the use of alternate promoters (see **PART 2** below) and reporters (see **PART 3 and 4** below).
3. To make the sensor more fieldable, the team also designed a 3D-printable and portable device that enables imaging of luminescence from a mobile phone. This device was designed to be used in conjunction with a mobile phone application for determining manganese chloride levels in the water sample(s) being tested. This work is described in the  [Wiki Implementation and Special Award: Hardware Wiki](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27hardware%27%29%20%7D%7D).