Wet Lab Enzyme Documentation

Overview

We will be utilizing a novel plasmid to generate the enzymes necessary for 7-methylxanthine synthesis. This involves combining 6x histidine-tagged NdmA (BBa_K4580000), 6x histidine-tagged NdmB (BBa_K4580001), 6x histidine-tagged NdmD (BBa_k4580002), and GFP (BBa_E0040) via Gibson assembly. NdmA and NdmB encode N-demethylases that will serve as our primary enzymes. NdmC, which is also present on the NdmABCD operon, was not utilized due to a knockout of the gene having little effect on caffeine demethylation as demonstrated by Dr. Ryan Summers [5]. GFP will allow for successfully transformed bacteria to be identified throughout our adaptive evolution cycle, and histidine tagging will facilitate protein purification in Ni-NTA affinity chromatography. Other components of the plasmid include two constitutive promoters (BBa_J23100 and BBa_J2310), a ribosome binding site (BBa_B0030), a terminator (BBa_B0010), and the plasmid backbone pSBC13. All parts listed can be found in the iGEM registry. The new basic parts created by Team Cornell will allow future teams to work with the enzymes separately rather than the full operon and gain increased flexibility and control within their projects involving the caffeine demethylation pathway.

Basic Parts

Name	Type	Description	Designer	Length (BP)
BBa_K4580000	Protein Coding Sequence	NdmA-6x his	Michael Constant	1075
BBa_K4580001	Protein Coding Sequence	NdmB-6x his	Michael Constant	1086
BBa_K4580002	Protein Coding Sequence	NdmD-6x his	Michael Constant	1785

Composite Parts

Name	Type	Description	Designer	Length (BP)
BBa_K4580004	Composite	NdmB-6x his-GFP	Michael Constant	1806

Wet Lab Protocol Manual

Basic Protocols

General Wet Lab Protocols

Gibson Assembly Protocol

High Efficiency Transformation Protocol for DH5ɑ strain of E.coli cells

Transformation Protocol for BL21(DE3) Competent Cell

2.5 mM LB + Caffeine (LBC) Media

Purpose: Protocol to make LB with caffeine

• Used for caffeine cultures and is the driving force behind all ADE (adaptive/directed evolution) experiments

Safety Considerations:

• PPE: gloves

Disposal:

• Dispose all used materials in appropriate waste containers

Storage:

• Store LBC media at room temperature

Materials:

• 20 g/L LB
• Caffeine
• 50 mL cone tube

Preparatory Steps:

• Make stock of 25 mg/mL LB

Method:

1. Heat 50 mL LB on hot plate on low with low stirring
2. Add ~25 mg caffeine and stir for 5 mins until dissolved
3. Label 50 mL cone tube and store at room temperature

NOTE: Adapt this protocol for different volumes and different concentrations of LBC

Media Change

Purpose: To change media of bacterial caffeine cultures

• Once a certain OD₆₀₀ (optical density) is hit, we need to change the media so it’s fresh
• This process also allows us to change media without losing biotic material

Safety Considerations:

• Perform all steps in BSC
• If transporting between BSC and elsewhere, all containers (eppendorf tubes, well plates) with biotic material must be CLOSED
• Gloves and lab coat

Disposal:

• All materials in contact with biotic material = biohazard waste
• If these contain liquid, neutralize liquid in separate container with bleach, DO NOT throw out liquids in biohazard waste
• Old media
• Neutralize with bleach
• Dump in sink

Storage:

• Well plate with new media in incubator at 37℃
• Store LB and LB/caffeine media in room temperature

Materials:

• LBC or LB media (depends on experiment being conducted)
• Centrifuge + centrifuge tubes (eppendorf tubes)
• OD₆₀₀ data
• Labeled waste container/beaker

Preparatory Steps:

• Prepare LB + caffeine (LBC media)
• Prepare BSC

Method:

1. Take OD₆₀₀ of each well and record in Benchling database
2. Label all eppendorf tubes with plate and well number
3. Transport well plate to BSC, maintain sterile technique
4. Completely transfer culture in each well into respective eppendorf tube
5. Centrifuge at 16000 rcf for 1 min
6. Quickly dump supernatant into waste beaker
1. Supernatant is the liquid portion (everything except for the bacterial pellet at the bottom of the tube)
7. Resuspend bacteria in 1.0 mL LBC or LB media
1. Pipette up and down to ensure pellet is broken down

Adaptive Evolution (AE)

Purpose: Set up and maintain an adaptive evolution experiment

Safety Considerations:

• PPE: wear gloves and lab coat
• All steps performed in BSC

Disposal:

• All materials in contact with biotic material = biohazard waste
• If these contain liquid, neutralize liquid in separate container with bleach, DO NOT throw out liquids in biohazard waste
• Old media
• Neutralize with bleach
• Dump in sink

Storage:

• Well plates in incubator at 37℃
• Store LB and LBC media in room temperature

Materials:

• LBC + LB media (modulate concentrations if needed)
• OD₆₀₀ data
• Labeled waste container/beaker
• Sterile 24 well plate

Preparatory Steps:

• Prepare LB + caffeine (LBC media)
• Prepare BSC (perform all steps in
• OD₆₀₀ + record data in Benchling database

Method:

**ONLY perform protocol if OD600 is between 0.5 - 0.8 (with breathing room)

1. Add 900 uL of LB or LBC media into new well plate
2. Repeat this cycle for each well
1. Pipette each sample up and down
2. Pipette 100 uL sample
3. Pipette into respective well in new plate
4. Record sample in each well in the Benchling database
3. Put in incubator at 37℃

Directed Evolution (DE)

Purpose: Generating a second round of mutations after the initial round to generate a more robust library of variants. Ensures that we don’t generate too many harmful mutations

Safety Considerations:

• MnCl₂, or Manganese (II) Chloride is organ toxic and toxic if swallowed or inhaled
• PPE: wear gloves
• Do not need to perform in BSC

Disposal:

• All materials in contact with biotic material = biohazard waste
• If these contain liquid, neutralize liquid in separate container with bleach, DO NOT throw out liquids in biohazard waste
• Old media/culture
• Neutralize with bleach
• Dump in sink

Storage:

• Well plates with new media in incubator at 37℃
• Store LB and LBC media in room temperature

Materials:

• PCR materials
• DNA cleanup materials
• Cell lysing solution and materials
• MnCl₂
• epPCR materials

Preparatory Steps:

• Ensure OD₆₀₀ is between 0.5 - 0.8 (or close enough)
• Ensure you have LB, LBC media
• Ensure you have lots of eppendorf tubes, spin columns, easy to track documentation
• Ensure you have enough material to perform lysing, DNA cleanup, and epPCR
• All steps can be performed on iGEM bench (no need for BSC)

Method:

1. Document OD₆₀₀ of each well in plate being used for DE
2. Lyse bacteria following cell lysis protocol
3. Perform miniprep, isolate plasmid
4. Perform epPCR
1. Nanodrop to ensure PCR took place
5. Transform BL21 bacteria and plate
6. Establish caffeine culture with plated colonies

Error Prone PCR (epPCR)

Purpose: Induce mutagenesis of DNA parts in controlled, high-throughput manner

• Introduces random mutations, useful for enzyme engineering
• Selective primer design provides specificity to region of DNA
• Changing concentration of DNA affects rate of mutation

Safety Considerations:

• PPE: Wear gloves

Disposal:

• Dispose all materials into appropriate waste containers

Materials:

• DNA Polymerase
• MnCl₂ and MnCl₂ solution

Preparatory Steps:

• Dilute DNTP to appropriate concentrations
• Dilute primers and DNA parts to appropriate concentrations
• Have Taq polymerase, sterile water and
• This template will help calculate the amount of uL for each step, edit to put in concentrations you want and what part you are doing

Method:

1. Make PCR Buffer
2. Utilize this table to make the 50 uL mixture for epPCR

Component	Volume
Nuclease Free Water	50 uL - ΣVother components
40 uM dATP	2 uL
40 uM dGTP	2 uL
40 uM dCTP	2 uL
40 uM dTTP	2 uL
Forward Primer	2 uL
Reverse Primer	2 uL
DNA template	2 uL
Q5 DNA polymerase	2 uL
MnCl2	[experimentally detrimented]

3. Program epPCR cycle on thermal cycler:

epPCR Modelling Results

Enzyme Immobilization - Alginate Bead Encapsulation

Purpose: Immobilization of enzymes within encapsulated calcium alginate beads

Safety Considerations:

• 0.8 mm needle is used
• Wear gloves
• Be careful with the needle (keep it facing away from you when not in use)

Disposal:

• Dispose needle in sharps box (found in BSC room in B07 on the back bench closest to microscope room)
• All pipette tips in normal tip waste

Storage:

• Store alginate beads at 4℃

Materials:

• Calcium Chloride, anhydrous
• Sodium Alginate
• Solution of enzyme of choice
• Stir plate (use hot plate with no heat, only light stirring)
• Stir bar
• Syringe
• 8-inch needle
• Filter paper
• Funnel

Preparatory Steps:

1. Prepare stock of 100 mL 1M calcium chloride (CaCl₂)solution
1. 11.098g of CaCl₂ in 100mL of distilled water
2. Prepare stock of 1.5% weight volume (w/v) sodium alginate solution with distilled water
1. i.e. for 1.5 g of sodium alginate, add 100 mL of distilled water
2. Instructions (DO NOT HEAT UP):
1. Add 100 mL of distilled water, put in stir bar and set to low
2. SLOWLY add a quarter of alginate
3. Then SLOWLY increase the spinning speed
4. Add the rest of the alginate
3. Identify the concentration of enzyme solution

Method:

1. Dissolve enzyme solution in sodium alginate solution to obtain 2 mg/mL concentration
2. Put 10 mL of 1 M CaCl₂ solution into a beaker
3. Attach needle to syringe, take up enzyme-alginate solution syringe, and add to CaCl2 solution dropwise
4. Incubate for 30 mins at room temp (stirring off)
5. Transform BL21 bacteria and plate
6. Collect beads by filtration, store at 4℃

For Automatic Bead Making:

1. Load the syringe into a syringe infusion pump
2. Set the infusion rate of the pump to 1mL/min
3. Set the 10mL of 1M CaCl2 under the tip of the syringe
4. Run the infusion pump while monitoring to ensure correct bead formation
5. Incubate the beads for 30 minutes at room temperature
6. Collect the beads by filtration and store at 4℃

Relevant Data:

The mean bead volume, the diameter of beads, and immobilization efficiency are all useful quantities to obtain

Mean Bead Volume:

Diameter of Beads:

Immobilization Efficiency:

Ni-NTA Affinity Chromatography

Buffer Preparation

Purpose: Preparation of relevant buffers for Ni-NTA affinity chromatography

Safety Considerations:

• Wear gloves
• When using imidazole, or MES buffer, YOU MUST work in a fume hood, YOU MUST wear goggles and a lab coat

Disposal:

• Dispose of all Ni-NTA chromatography buffers in a new waste container called HisPur waste (ONLY OPEN IN FUME HOOD)
• If the waste container becomes full, let Adi or Michael know and we will dispose of it

Storage:

• MES buffer: 4C fridge
• Imidazole: room temperature
• PBS: room temperature

Materials:

• PBS (phosphate buffered saline)
• Imidazole
• MES buffer (2-(N-morpholine)-ethanesulfonic acid)
• Sodium chloride solution

Preparatory Steps:

• Make all buffers in 100 mL volumes (so we don’t waste material)

Equilibration/Binding Buffer (10 mM imidazole, pH 7.4):

1. In 250 mL of PBS, add 172 mg of imidazole, mix until dissolved
2. Label as E/B buffer with date, store in room temperature

Wash Buffer (25 mM imidazole, pH 7.4):

1. In 250 mL of PBS, add 430 mg of imidazole, mix until dissolved
2. Label as wash buffer with date, store in room temperature

Elution Buffer (300 mM imidazole, pH 7.4):

1. In 100 mL of PBS, add 2.06 g of imidazole, mix until dissolved
2. Label as elution buffer with date, store in room temperature

MES Buffer (pH 5.0):

1. In 250 mL PBS, add 344 mg of MES, mix until dissolved
2. Label as MES buffer with date, store in room temperature

Purification
Purpose: Separating out his-tagged compounds from bacterial lysate

Safety Considerations:

• Wear gloves
• When using imidazole, or MES buffer, YOU MUST work in a fume hood, YOU MUST wear goggles and a lab coat

Disposal:

• Dispose of all Ni-NTA chromatography buffers in a new waste container called HisPur waste (ONLY OPEN IN FUME HOOD)
• If the waste container becomes full, let Adi or Michael know and we will dispose of it

Storage:

• E/B, wash, and elution buffers can be stored in room temperature

Materials:

• E/B, wash, elution buffers
• Eppendorf tubes
• Nanodrop
• Syringe
• 0.2 µm syringe filter
• 15 mL Falcon tubes
• pH strips
• Beaker

Procedure:

Step 1: Equilibrate Ni-NTA cartridge

1. After lysing bacteria and collecting supernatant, sterile filter supernatant, collect supernatant in Falcon tube
2. Remove top plug from cartridge and carefully SNAP off end-tab
1. DO NOT TWIST the end-tab
3. Equilibrate cartridge with 5-10 column volumes (5-10 mL) of E/B buffer at a flow rate of 1-2 mL/min
1. Use a syringe to inject buffer into column
2. Rule of thumb: 30 drops per minute = 1 mL/min flow rate

Step 2: Prepare sample

1. Mix filtered lysate 1:1 with E/B strength
1. Ensure pH is around physiological pH (7.4)
2. Use a new syringe to inject mixed sample to cartridge
1. Inject at a rate of 0.5-1 mL/min (15-30 drops/min)
2. Collect flow through in a waste beaker
3. Wash column with 10-15 mL of wash buffer at 1 mL/min
1. Collect flow through in waste beaker

Step 3: Elute compound of interest

1. Use 5-10 mL of Elution Buffer at flow rate of 0.5 mL/min to collect fractions in 1 mL eppendorf tubes
1. Number the tubes
2. Wash cartridge with five mL of diH2O (1 mL/min flow rate)
3. Fill entire column with 20% ethanol, close it properly

Step 4: Identify/Quantify Compound of Interest

1. Use nanodrop calibrated for protein concentration and measure concentration in each tube

Step 5: Regenerate Ni-NTA resin

1. Wash cartridge with 10 mL of MES buffer
2. Wash cartridge with 10 mL of diH2O
3. Store the cartridge with 20% ethanol in it.

Before reuse, re-equilibrate with E/B buffer before continuing with step 1 of the protocol
(Inspired by ThermoFisher HisPure Ni NTA Chromatography protocol)

Product Development Protocol Manual

Bioreactor Assembly

Purpose: Assembly of a bioreactor that immobilizes sodium alginate beads, pumps reactant with caffeine molecules through, and provides enough residence time to synthesize seven-methylxanthines.

Safety Considerations:

• During assembly, no PPE is needed.
• During operation, wear gloves.

Disposal:

• Enzyme beads can be reused. If they do need to be replaced,the beads can be disposed of with general waste.
• All other materials should only be replaced and disposed of if broken. These can also be disposed of with general waste.

Materials:

• L298N Motor Drivers
• Jumper cables/electrical wires
• Arduino Mega/Uno (depending on the number of motors you are wiring)
• 12V Power supply
• Arduino USB cable
• Bead immobilization lattice (CAD File)

How to build: Link

Encapsulation Protocol

Policy and Pratices

EUDI

Last year we began to develop an ethical framework for developing and evaluating a project called EUDI, or Empathize, Understand, Develop, Implement and Assess. This year we decided to develop it further by getting feedback from stakeholders and various ethics experts in the field. Our hope is that future iGEM teams can take EUDI and apply it to their project in a similar manner to us, focusing on how to best help their communities in an ethical and effective manner.

Ethics Case Study Slideshow

This year we hosted various bioethics debates at local nursing and retirement homes in an effort to encourage discussion between us and the community and in an effort to gain different perspectives. We have linked the slideshow above that includes one case study of our own project which other teams can replace with their project and two more than target case studies that were understandable by people with little to no science background. We hope that other teams can use such a slideshow and use it as a model for a similar outreach project they have.

Outreach Lesson Plan for Younger Children

We have linked the outline for a lesson plan that Cornell iGEM developed in order to engage younger students with synthetic biology and biology in general through hands-on activities. We did include one activity that was specific to our project for the year which teams can adapt to their specific projects. We wanted to use materials and methods that were inexpensive and accessible, other teams may use this lesson plan to develop activities for their own outreach events.

Biobuilder

We decided that a great way to encourage younger students to get involved and learn more about synthetic biology would be in the form of a game. An online game like BioBuilders is easily accessible by people all across the world. We built the game as a way for children to learn about the process behind developing a synthetic biology project using various iGEM projects over the years, including ENERGEM. We hope that other teams can use this game at any outreach event they use as a way to help educate children about synthetic biology. They can also develop similar games, adding with their own projects, leading to a giant network of projects that we can all contribute to within this game.

Science Experiment Video Series

This year, we decided to create a video series for children that focused on teaching them important science concepts related to iGEM and our project. We wanted to be as engaging as possible and interactive so we filmed a series of experiments, the majority of which people can do from their home. We wanted them to be as accessible as possible, which is why we filmed them online. We had 4 total videos, focusing on topics ranging from DNA, polymer chemistry, to 7-methylxanthines and released them throughout the end of the summer and the fall. We hoped that this would be an opportunity for other teams to use in their outreach efforts or expand on these videos with experiments of their own.

References

[1] Copp, J. N., Hanson-Manful, P., Ackerley, D. F., & Patrick, W. M. (2014). Error-Prone PCR and Effective Generation of Gene Variant Libraries for Directed Evolution. In E. M. J. Gillam, J. N. Copp, & D. Ackerley (Eds.), Directed Evolution Library Creation (Vol. 1179, pp. 3–22). Springer New York. https://doi.org/10.1007/978-1-4939-1053-3_1

[2] Fromant, M., Blanquet, S., & Plateau, P. (1995). Direct Random Mutagenesis of Gene-Sized DNA Fragments Using Polymerase Chain Reaction. Analytical Biochemistry, 224(1), 347–353. https://doi.org/10.1006/abio.1995.1050

[3] Ling, L. L., Keohavong, P., Dias, C., & Thilly, W. G. (1991). Optimization of the polymerase chain reaction with regard to fidelity: Modified T7, Taq, and vent DNA polymerases. Genome Research, 1(1), 63–69. https://doi.org/10.1101/gr.1.1.63

[4] Random Mutagenesis by PCR - Wilson—2000—Current Protocols in Molecular Biology—Wiley Online Library. (n.d.). Retrieved August 8, 2023, from https://currentprotocols.onlinelibrary.wiley.com/doi/10.1002/0471142727.mb0803s51

[5] Summers, R. M., Louie, T. M., Yu, C. L., Gakhar, L., Louie, K. C., & Subramanian, M. (2012). Novel, highly specific N-demethylases enable bacteria to live on caffeine and related purine alkaloids. Journal of bacteriology, 194(8), 2041–2049. https://doi.org/10.1128/JB.06637-11

[6] Team:Cornell—2019.igem.org. (n.d.). Retrieved August 8, 2023, from https://2019.igem.org/Team:Cornell

[7] Urrea, D. A. M., Gimenez, A. V. F., Rodriguez, Y. E., & Contreras, E. M. (2021). Immobilization of horseradish peroxidase in Ca-alginate beads: Evaluation of the enzyme leakage on the overall removal of an azo-dye and mathematical modeling. Process Safety and Environmental Protection, 156, 134–143. https://doi.org/10.1016/j.psep.2021.10.006

[8] Wong, T. S., & Tee, K. L. (2020). Gene Mutagenesis. In T. S. Wong & K. L. Tee, A Practical Guide to Protein Engineering (pp. 121–148). Springer International Publishing. https://doi.org/10.1007/978-3-030-56898-6_8