iGEM 2023 — Team okstate

January

* Recruit Members
* Research past iGEM projects
* Develop Project Plan

February

* Recruit Members
* Research past iGEM projects
* Develop Project Plan
* Begin fundraising

March

* Recruit Members
* Research past iGEM projects
* Develop Project Plan
* Continue Fundraising

April

* Research past iGEM projects
* Develop Project Plan
* Begin Plasmid Design
* Begin Enzymology Experiments
* Begin Dry Lab development

June

* Work on Safety Form
* Continue Cloning Lab work
* Continue Enzymology Experiments
* Continue Dry Lab Experiments

July

* Continue Cloning Lab work
* Continue Enzymology Experiments
* Continue Dry Lab Experiments

August

* Continue Cloning Lab work
* Continue Enzymology Experiments
* Continue Dry Lab Experiments

September

* Visit OSU Wastewater Treatment Plant
* Visit Stillwater Middle School
* Work on Promotional Video
* Work on Wiki

October

* Finale Data/Results
* Work on Wiki
* Make Project Presentation

November

* iGEM Grand Jamboree!

Research

Generally when people talk about the engineering design cycle, they start with “Design”, but that skips over a crucial part of the design process, that being research. Before attempting to design our own fluorometer we needed to further our understanding of how fluorometers actually worked. To do this we used online resources to educate ourselves on what parts are needed for a functioning fluorometer and did research on what other IGEM teams have done in the past when making similar hardware.

Design

We took our basic understanding of fluorometers and applied them to make a design that we believed would work in a lab setting, prioritizing low cost, ease of manufacturing, and functionality specifically for 7-hydroxycoumarin. Over the course of a few brainstorming meetings, we decided on an initial design that was simple but also theoretically effective.

Build

We then used solidWorks to create a 3-D model of our first fluorometer design, along with models for some of the other essential parts for function. These models can be seen below.

![](https://static.igem.wiki/teams/4884/wiki/engineering/image1.png)

![](https://static.igem.wiki/teams/4884/wiki/engineering/image4.png)

Test

Due to difficulty acquiring materials, we were never able to build a physical model of our fluorometer and therefore couldn’t run tests in the classical sense. Instead, we took our design and sought input from the other students and advisors on the team to find ways to improve our design. Taking input from different perspectives allowed us to locate flaws in our design that we may not have considered, which could then be fixed in later design iterations.

Learn

The primary concerns with our initial design were light pollution from the LED potentially getting through the UV film, and an inability to use the fluorometer in a non-lab environment. We took these concerns and brainstormed ways we could improve our design.

Design

Using the concerns brought to us regarding our previous design, we redesigned the layout of the fluorometer and created an autonomous system for switching samples for use in a real world setting relevant to our project.

Build

A new 3-D model was made for our redesigned fluorometer in SolidWorks.

![](https://static.igem.wiki/teams/4884/wiki/engineering/image3.png)

![](https://static.igem.wiki/teams/4884/wiki/engineering/image2.png)

Test

Like with our first model, we opted to seek out input from the other members of our team. This time, there were no major concerns for our design.

Learn

Through a full iteration of the engineering design cycle, we were able to come to a design that we believed would fulfill the needs of our project. While there are likely more improvements that could be made through further iterations of the design cycle, we decided through our method of testing that our second design was sufficient.

General Protocols

1. LB broth and agar preparation

* To make liquid broth, we dissolved commercially available LB powder (Fisher Scientific) into ddH2O according to the manufacturer's recommended instructions.
* To make LB agar, we prepared the same amount of the LB powder as above and added sufficient agar powder (Fisher Scientific) to make a final concentration fo 1.5%.
* Mixtures were prepared in glass bottles with caps and autoclaved.
* When appropriate, antibiotics were added into the LB before bacterial culture. For LB agar, antibiotics were added prior to pour plate.
* Prepared media were stored in sealed plastic bags inside a 4C refrigerator until use.

2. Nitrate minimal salts (NMS) medium preparation

* Nitrate minimal salts medium were prepared according to the table below
* After all ingredients below were added, 700ml of water were added to dissolve, then water were added up to 1 liter. Medium were then autoclaved.

|  |  |
| --- | --- |
| Ingredient | Amount per liter |
| MgSO4•7H2O | 1.0 g |
| KNO3 | 1.0 g |
| CaCl2•H2O | 0.2 g |
| 3.8% (w/v) solution Fe-EDTA | 0.1 ml |
| 0.1% (w/v) NaMo•4H2O | 0.5 ml |
| Trace element solution (recipe below) | 1.0 ml |
| Bacto-agar (if making plates) | 15 g |

* Trace element solution (1X) were made according to the table below. All components were added to 700ml of water to dissolve then add water up to 1 liter. Solutions did not need to be sterilized.

|  |  |
| --- | --- |
| Ingredient | Amount per liter |
| FeSO4•7H2O | 500 mg |
| ZnSO4•7H2O | 400 mg |
| MnCl2•7H2O | 20 mg |
| CoCl2•6H2O | 50 mg |
| NiCl2•6H2O | 10 mg |
| H3BO3 (boric acid) | 15 mg |
| EDTA | 250 mg |

* After the above were made and autoclaved, 10ml per liter of autoclave phosphate stock (see below) and 10ml per liter of filter-sterilized vitamin stock (see below) were added.
* Make 1 liter solution of the below stock. pH to 6.8 before autoclaving.

|  |  |
| --- | --- |
| Phosphate stock solution (1x) | |
| Ingredient | Amount per liter |
| KH2PO4 | 26 g |
| Na2HPO4•7(H2O) | 62 g |

* Make 1 liter of the vitamin stock and sterilize by using a 0.2micron filter.

|  |  |
| --- | --- |
| Vitamin Stock (1X) | |
| Ingredient | Amount (per liter) |
| Biotin | 2.0 mg |
| Folic acid | 2.0 mg |
| Thiamine HCl | 5.0 mg |
| Ca pantothenate | 5.0 mg |
| Vitamin B12 | 0.1 mg |
| Riboflavin | 5.0 mg |
| Nicotiamide | 5.0 mg |

* Lastly, CuCl2-2H2O were added to a final concentration of 10micromolar (1.4mg/liter) to complete the medium.

3. MOPS buffer

* Ingredients below were added along with 800ml of dd water. pH were adjusted to 7.0 and then water were added up to 1 liter.

|  |  |  |
| --- | --- | --- |
| Ingredients | Amount per liter | Concentration |
| MOPS | 41.86g | 0.2M |
| Sodium acetate | 4.1g | 0.05M |
| Disodium EDTA | 3.72g | 0.01M |

4. E. coli culturing

* E. coli strains were routinely streaked out from frozen glycerol stocks onto LB agar plates containing appropriate antibiotics (100 microgram per mil Ampicillin and/or 20microgram per mil chloramphenicol). Single colonies were picked with sterile tips or toothpicks and inoculated into LB broth containing appropriate antibiotics. Culture were grown at 37C in a shaking incubator (200 rpm).

5. Making E. coli competent cells

* TFB1 buffer were prepared by combining all ingredients below. pH were adjusted to 5.8 using 0.2% acetic acid. Buffer were filter sterilized and stored at room temperature.

|  |  |
| --- | --- |
| Ingredients | Concentration |
| Rubidium chloride | 100mM |
| Manganese chloride | 50mM |
| Potassium Acetate | 30mM |
| Calcium chloride | 10mM |
| Glycerol 15% |  |

* TFB2 buffer were prepared by combining all ingredients below. pH were adjusted to 6.5 using KOH.. Buffer were filter sterilized and stored at room temperature.

|  |  |
| --- | --- |
| Ingredients | Concentration |
| MOPS | 10mM |
| Rubidium chloride | 10mM |
| Calcium chloride | 75mM |
| Glycerol | 15% |

* E. coli strains were cultured according to above description until log phase growth, culture were kept on ice until use
* Cells were concentrated by centrifugation at 5000g for 10 min (at 4C). Supernatants were discarded and 100ml of TFB1 were added per 250mL of growth culture and resuspended.
* Mixture were incubated on ice for 5 min and then concentrated via centrifugation at 5000g for 5 min. Supernatant were discarded.
* 10ml of TFB2 were added to the cell pellet and gently resuspended. After incubating on ice for 60 minutes, 100 microliter of the mixture were aliquoted into pre-chilled 1.5mL microcentrifuge tube and snap freeze.
* Competent cells were stored at -80C freezer.

6. Plasmid transformation

* 2 microliter of purified plasmid DNA were added into 1 vial of competent cell containing up to 100 microliter. DNA were gently mixed with sterile pipet tips and the mixture were incubated on ice for 30 minutes.
* With the cap closed, the tube were dipped into water pre-heated to 42 degrees celsius for 1 min, and then immediately placed back on ice for 1 min.
* 500ml of LB broth were then added to the mixture and the tube were incubated at 37C with shaking (200rpm) for 1 hour.
* After centrifugation (10,000RPM for 1 min), 400 microliters of the supernatant were removed and the remaining suspension were plated into LB agar plates

7. Plasmid extraction

* Plasmid extraction were performed according to the instruction provided from the manufacture. We used the Thermo Scientific GeneJet Plasmid Miniprep Kit for all plasmid extraction.
* Briefly, E. coli strains carrying our plasmid were cultured in LB broth overnight and next day 2ml of the culture were collected by centrifugation at 10,000RPM for 1 min. Cell pellet were collected by discarding the supernatant.
* We then proceeded to extract the plasmid according to the provided instruction with the exception that at the end we eluted plasmid DNA using 50microliter of sterile water. DNA concentration were measured using the NanoDrop ND-1000 UV-Vis Spectrophotometer. DNA are stored at -20C.

8. SDS-Polyacrylamide Gel Electrophoresis

* To prepare a 10% resolving gel, we prepare 5ml of solution containing 1.3ml of 1.5M Tris-HCl at pH 8.8, 50 microliter of 10% w/v SDS, 1.275ml of 29:1 acrylamide mix, and add water up to 5mL.
* To polymerize the resolving gel, we added 4 microliter of TEMED and 50 microliter of 10% ammonium persulfate (APS). The resolving gel was loaded into a gel casting stand containing two glass cassettes. Sterile water were then added to overlay the resolving solution to prevent evaporation. We typically wait about 20 to 30 minutes for the gel to polymerize.
* To prepare the 5% stack gel, we prepared 2 ml of solution containing 0.25mL of 1.0M Tris-HCl at pH 6.8, 20 microliter of 10% w/v SDS, and 0.2475ml of 29:1 acrylamide mix. 2 microliter of TEMED and 20 microliter of 10% APS were then added to facilitate polymerization.
* The stacking solution were added on top of the polymerized resolving gel, a comb was placed over the top and we typically wait for 30 minutes for full polymerization.
* To prepare E. coli cell lysates for protein separation, 1ml of overnight bacterial culture would be concentrated via centrifugation at 10,000rpm for 1 min, the cell pellet would then be dissolved in 5 microliter of 5X SDS sample buffer containing 1M Tris-HCl at pH 8.0, 50% w/v glycerol, 10% w/v SDS, 1% w/v bromophencol blue, and 12.5 v/v of beta-mercaptaethanol to a final volume of 25 microliter (in PBS buffer). Protein samples would be heated at 97C for 20 minutes and then kept on ice.
* Next, 5 microliter of the mixture were loaded into the gel along with a pre-stained protein ladder (Fisher-Scientific). Electrophoresis were performed at 100 volts until the dye front has reached the bottom. Electrophoresis were performed with 1X running buffer (25mM Tris-base, 192mM glycine, 0.1% w/v SDS, pH 8.3) using the Bio-Rad Mini-Protean Tetra gel system.
* Protein bands were visualized by staining the gel in 100ml of the staining solution (to make 1 liter combine 1 g Coomassie R250 to 300ml methanol, 50ml acetic acid, and 650ml of water. The gel can be destained by soaking in destaining solution (10% acetic acid, 50% methanol, and 40% water) for at least 2 hours until the background are clear.

Measurement of sMMO activity by coumarin

1. Culturing of M. trichosporium

* M. trichosporium were kindly provided by Dr. Babu Fathepure (OSU)
* M. trichosporium were inoculated into 30ml of NMS broth maintained in 125-ml serum vials. Syringe was inserted into the rubber septa to contain 50% methane and 50% air. The culture were incubated at 30C with shaking.
* 48 hours post-incubation, cell culture were harvested and washed 3 times with 20mM MOPS buffer by centrifugation at 5,000g for 5 min.
* After discarding supernatant, cells were resuspended in MOPS buffer containing 5 mm MgSO4 and 20mm sodium formate. Cell suspension were kept on ice.

2. Preparation of coumarin for enzyme activity

* To generate a standard curve, Coumarin purchased from Sigma Aldrich and dissolved in 20mM MOPS buffer at the following final concentration: 700mM, 70mM, 7mM, 0.7mM, 0.07mM, 0.007mM, and 0.0007mM.
* To measure sMMO activity, coumarin were prepared in final concentration of 0.5mM.

3. Enzyme activity detection

* 5ml of cell suspensions prepared in step 1 were mixed with 20mM sodium formate and appropriate volume of coumarin and sealed in glass serum vials for 1 hour at 30C with shaking.
* Reactions were stopped by filtering out the cells using 0.8 micron pore-size syringe filters followed by 0.2 micron pore-size syringe filters.
* Fluorescence were determined using the PTI QuantaMaster fluorescence spectrometer with excitation at 338nm and emission at 450nm.

Introduction

Anaerobic digestion of dairy farm waste products such as wastewater and biosolids into useful energy-rich has been implemented in large farms throughout the world. However, the biogas derived from such systems, although have been shown to reduce methane emission, are not easily collected due to inefficient gas to liquid conversion within the system. Researchers have proposed the addition of methanotrophic bacteria as a possible solution due to their ability to utilize methane as a carbon energy source. However, maintenance of the methanotrophic bacteria are difficult and not cost-effective.  Therefore, by taking inspiration from the 2014 Braunschweig and 2021 Wageningen teams, we wished to engineer an E. coli strain capable of expressing the soluble methane monooxygenase enzyme (sMMO) from Methylococcus capsulatus. sMMO can catalyze the conversion of methane into methanol, which can then be readily extracted as energy source.

Construct design

The biobrick design of the 2014 Braunschweig team inspired us to adapt their design. The biobrick (BBa\_K1390019) designed by the team included all 6 genes that encodes the different subunits of the soluble MMO. The construct was placed into pSB1A3 and transformed into E. coli JM109. In addition, the Braunschweig team co-transformed two chaperones GroEL and GroES using Takara Clontech’s Chaperone Plasmid C2. (see below for schematic of the biobrick design).

![](https://static.igem.wiki/teams/4884/wiki/results/image1.png)

In our design, we decided to create several modifications that we believe can increase the expression and therefore activity level of the soluble sMMO:

1. Codon optimization. In the original design, the mmo genes were obtained from the genome of M. capsulatus. We decided to optimize all 6 ORFs to codons suitable for E. coli expression. With this design, we created BBa\_K4884006 (see below).

![](https://static.igem.wiki/teams/4884/wiki/results/image3.png)

2. Promotor optimization. In the second design, we decided to swap out the promoter with the inducible AraBAD promoter. We believe this stronger promoter might also enhance expression. Furthermore, since the chaperone carrying plasmid from Takara Biotech also uses an arabinose inducible promoter, this design would allow us to induce both the sMMO subunits as well as the chaperones. With this design, we created BBa\_K4884006 (see below).

![](https://static.igem.wiki/teams/4884/wiki/results/image4.png)

Results:

Due to time constraints and also funding issues, we decided to submit the two biobrick constructs to IDT for full synthesis. Since we codon optimized all the mmo genes, this was really the only way. In addition, we decided to request IDT to clone the entire construct into their pUC57 based plasmids which encodes for ampicillin resistance. We were successful in obtaining the plasmids from IDT.

We next transformed the two plasmids into E. coli DH5alpha and selected on Amp 100 LB agar plates. After obtaining single colonies, we made glycerol stocks. We also were successful in transform the Takara chaperone Plasmid C2 into these two E. coli strains and selected for both plasmids on LB agar containing ampicillin and chloramphenicol.

Unfortunately, due to time constraints we were unable to test the expression of the sMMO enzyme in our system.

Development of a rapid fluorescent based assay to detect sMMO activity

A critical assay for any team that works with sMMO would be to confirm the activity of the sMMO enzyme. Namely, whether it has successfully converted methane into methanol. The 2014 Braunschweig team created an anaerobic jar in which methane concentration was detected via a MQ-4 semiconductor sensor. In our literature search, we identified another potentially cheaper alternative. In a 2002 publication by MIller et al, they discovered that coumarin can be oxidized in the presence of a functional sMMO into 7-hydroxycoumarin. The emission spectrum of 7-hydroxycoumarin is significantly different from that of coumarin to be detected and quantified using a fluorometer.

![](https://static.igem.wiki/teams/4884/wiki/results/image2.png)

Results:

Based on the protocol described by Miller et al, we were able to purchase coumarin and obtained Methylosinus trichosporium cell lysates. Unfortunately, the only fluorometer that was accessible to us at OSU broke when we attempted to use it, and we were unable to find another alternative.

Summary and future directions:

* We successfully obtained our new sMMO expressing plasmid and transformed them into E. coli DH5alpha along with plasmid C2 from Takara Biotech carrying E. coli GroEL and GroES.
* We ran out of time to test our E. coli strains for expression of the subunits.
* We devised a different way of measuring sMMO activity based on the oxidation of coumarin ino a fluorogenic substrate 7-hydroxycoumarin. All raw materials were successfully obtained, unfortunately due to equipment failure we were not able to set up the assay.
* Being the first ever iGEM team developed at OSU, we worked hard in forming a talented team of students and we were able to obtain funding so we could enter the competition. We wished we had more time and better luck in testing our E. coli strains. However, this process is now ongoing in our team.
* Once we have confirmed the activity of sMMO in our engineered E. coli, we would like to go a step further in building a prototype of our own [fluorometer](https://www.google.com/url?q=https://2023.igem.wiki/okstate/hardware&sa=D&source=editors&ust=1697101801640176&usg=AOvVaw3u4bOPGBnrKCo4G3ww2l3U) to create a mockup of what we envision our engineered bacteria can do in real life applications.
* We would also like to mutate the amino acid residues of the sMMO subunits in an attempt to increase enzyme activity. A result publication by Bennett et al (2021) performed directed evolution and identified many mutations that resulted in higher activity. We would like to repeat some of the experiments to see if it works in our case.

References:

[https://2014.igem.org/Team:Braunschweig](https://www.google.com/url?q=https://2014.igem.org/Team:Braunschweig&sa=D&source=editors&ust=1697101801640644&usg=AOvVaw2U6rF_paoZiRf2FRrWGOpg)

[https://2021.igem.org/Team:Wageningen\_UR](https://www.google.com/url?q=https://2021.igem.org/Team:Wageningen_UR&sa=D&source=editors&ust=1697101801640915&usg=AOvVaw27FKYVDLjqZh9zbMGgFv02)

Miller, A., et al. "A rapid fluorescence-based assay for detecting soluble methane monooxygenase." Applied microbiology and biotechnology 58 (2002): 183-188.

Bennett, R. Kyle, et al. "Expression of soluble methane monooxygenase in Escherichia coli enables methane conversion." BioRXIV (2021): 2021-08.