iGEM 2023 — Team uconn

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{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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# Project Notebook

# 2022

June

Literature review and project development was the main goal. Read articles and consulted previous iGEM projects on bacterial vs. fungal laccases. Created a list of potential bacterial laccase candidates based on other uses in wastewater pollutant degradation. Began weekly graduate advisory board meetings for discussion. Checked the stability of current lab reagents and created new LB Broth, 1x TAE buffer, and Ampicillin LB Agar plates. Settled on a list of four different laccases to utilize: *Bacillus subtilis, Bacillus licheniformis, Kocuria palustris, Paenibacillus glucanolyticus*.

July

Initially began with *B. subtilis* bacteria obtained from team advisor Charles Bridges. Completed genomic DNA extraction using the Qiagen DNA extraction kit. Designed primers for PCR isolation of the CotA laccase of *B. subtilis* followed by gel electrophoresis checks. Obtained pET22b(+) vector with an existing Profilin insert from the UConn Heaslip laboratory. Ran digestions of the vector using EcoRI and XbaI to cut out previous Profilin insert. Attempted a PCR extension of CotA to flank laccase sequence with EcoRI and XbaI.

August

Created a document of NCBI sequences for each of the four bacterial laccases with EcoRI on the 5' end and XbaI on the 3' end. Two coding sequences were created: one with an NSP4 signal peptide attached to the laccase and one with only the lone laccase gene. Used the IDT codon optimization tool to optimize sequence for *E. coli* expression and sent to IDT for gBlock synthesis.

Fall 2022

Attempted to remove the Profilin gene from the Pet22b plasmid by digesting with EcoRI and Xbal. Had difficulty with gel extraction and purification of digested vector. Transformations following ligation did not appear to result in well-isolated colonies. Other digestion attempts demonstrated that PCR reagents worked, but insert was of incorrect size.

# 2023

June

Overviewed project idea from previous year. Determined where supplies were in the lab and created an inventory of chemical reagents & equipment. Attempted to remove the Profilin insert from the donated PET-22b plasmid, with difficulty recovering DNA. Started to design Wiki.

July-August

Started to use ordered pET-22b(+) plasmid from company Novagen and to ligate one of our *Bacillus subtilis* IDT gBlocks into the vector after digestion. We started to screen colonies by PCR with the T7 primers that are found in the PET22b plasmid, but did not have one with the correct insert size. We started to use the gblock from *Bacillus licheniformis.*

September-October

We continued to digest the plasmid and the *Bacillus licheniformis* gblock. PCR screening by gel check indicated one colony that had an insert large enough to potentially be the gblock. We purified the PCR, set up cultures for glycerol stocks and minipreps, and sent the purified samples to be sequenced by Sanger Sequencing.

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{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
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#### Silver Medal Criterion #1

Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.

If you plan to show engineering success by creating a new Part that has been shown to work as expected, you must document your contribution on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page) for your team to be eligible for this criteria.

---

Please see the [2023 Medals Page](https://competition.igem.org/judging/medals) for more information.

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{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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# **Protocols**

# *LB Agar Plates*

In order to conduct transformation experiments, you need to create agar plates that have antibiotics to select the bacteria which successfully take up your plasmid. This protocol explains how we create agar plates.

**Materials**

* Tryptone
* Yeast Extract* Sodium Chloride
  * Agar
  * Stock Solution Antibiotic
  * Stirring plate and magnetic stirrer
  * Petri dishes (90mm)

**Procedure: Pretty Plates**

1. In a large 1L bottle: Add 5g tryptone, 2.5g Yeast Extract, 5g NaCl, 7.5g Agar, and 475mL dH2O.
2. Swirl or put on stir plate with autoclave-safe magnetic stir bar.
3. Make sure bottle cap is loose and autoclave on liquid cycle for approx. 1 hour.
4. Add appropriate concentration of antibiotic once bottle is cooled.

* For chloramphenicol: Add to final concentration of 35 ug/mL.
* For ampicillin stock: Add to a final concentration of 50 ug/mL final.

5. Pour the liquid agar into the plates one at a time, for a depth of about 1/4 inch. About 12-16 mL.
6. Wait about 1 hour for plates to solidify and then flip over. Dry for several hours to overnight at room temperature and store at 4C.

# *Polymerase Chain Reaction*

**Materials**

* Taq Polymerase
* goTaq Buffer
* Forward and Reverse Primer
* MgSo4
* Nuclease-free water
* dNTPs

**Procedure: Double or Nothing**

1. Create a Mastermix for the PCR reaction. Begin with 1x (one reaction) and multiple reagent amounts as needed. Double mastermix reagents to include a negative control. For one 25 ul reaction:

* 16.8uL dH2O
* 5uL GoTaq Buffer
* 0.7uL dNTPs
* 0.25uL GoTaq enzyme
* 0.25uL MgSO4
* 0.5uL Forward Primer
* 0.5uL Reverse Primer

2. Add 24 uL of the mastermix to one experimental and one negative control PCR Tube.
3. Add 1uL of DNA to the experimental PCR reaction tube and 1ul of water to the negative control PCR tube.
4. Place tubes in thermocycler.
5. Run reaction at:

* Initial Denaturation - 95C for 2 minutes - 1 cycle
* Denaturation - 95C for 1 minute - 25 cycles
* Annealing - 65C for 1 minute - 25 cycles
* Extension - 72C for 1 minute/kb - 25 cycles
* Final Extension - 72C for 5 minutes - 1 cycle
* Storage - 4C for infinity

6. Check results by running a gel

# *Digestion*

**Materials**

* Restriction Enzymes
* NEB CutSmart Buffer
* Plasmid or PCR DNA
* dH2O

**Procedure: Cutting Up The Pieces**

1. Calculate the amounts of each material needed for a 25uL reaction using 2.5uL of CutSmart Buffer, 0.5uL of each restriction enzyme, and volume of DNA based on nanodrop concentration. Utilizing 2-4ug of DNA is considered good for a digestion.
2. Thaw CutSmart Buffer and DNA. Make sure to keep restriction enzymes on ice at all times.
3. Pipette DNA and dH2O into 1.5 mL low adhesion tube.
4. Pipette 2.5 uL CutSmart buffer.
5. Add 1uL total of restriction enzyme. (0.5uL each if performing double digest)
6. Pipette gently to mix. Do not vortex.
7. Place reaction in an incubator or thermocycler at 37 C for 2-4 hours.
8. Run a gel to check results

# *Gel Electrophoresis*

**Materials**

* 1x Tris-Acetate EDTA (TAE) Buffer
* Agarose
* SYBR Safe for Band Visualization
* 10kb Ladder
* 6x concentrate purple loading dye
* Gel rig

**Procedure: Making Jelly and Checkin' Bands**

1. Dilute 50x concentrated stock TAE to make 1L of 1x TAE Buffer.
2. Measure 30mL 1x TAE in a clean erlenmeyer flask using a graduated cylinder.
3. Add 0.3g agarose to make a 1% gel.
4. Microwave for a maximum of 1:30 min until completely dissolved. Swirl every 10 seconds with hot hands.
5. Allow the mixture to cool in the flask until warm to the touch.
6. Add 3uL of SYBR Safe into the flask and swirl gently to mix.
7. Add comb to the gel rig and pour contents of the flask into the clean, dry mould.
8. Allow gel to harden. Remove comb once hardened.
9. Prepare samples by adding 1uL of purple loading dye to 5uL of sample DNA. For the ladder, add 4uL of dH2O + 1uL ladder + 1uL loading dye into a tube.
10. Fill the clean gel rig with 1x TAE buffer.
11. Load samples carefully, making note of what each well contains.
12. Run gel at 100V for 20 minutes. Use the dye front to determine migration of samples.
13. Image gel using UV light.

# *Ligation*

**Materials**

* Digested Backbone
* Digested DNA insert
* T4 DNA Ligase
* T4 DNA Ligase Buffer

**Procedure: How To Put Things Back Together**

1. Calculate the amount of backbone and insert DNA needed for the process based on the individual insert lengths and concentration determined from nanodrop. A ratio of 5:1 or 3:1 usually works best.
2. Considering the addition of 2uL of T4 Ligase Buffer and 1uL of T4 Ligase, calculate the amount of dH2O necessary for a 20uL total reaction.
3. Keep T4 ligase enzyme on ice until it needs to be added.
4. Add all components in the following order to a 1.5 ml microcentrifuge tube: dH2O, T4 Buffer, DNA Backbone, DNA insert, T4 Ligase enzyme.
5. Mix by pipetting up and down, and briefly spin to get contents to the bottom of the tube.
6. Incubate for 10 minutes at room temperature.
7. Heat inactivate at 65 C for 20 minutes.
8. Proceed to transformation or store reaction at -20C.

# *Transformation*

We utilize a modified version of the New England Biolabs High Efficiency Transformation Protocol

**Materials**

* NEB 5-alpha Competent *E. coli* cells
* Ligation Mixture
* Water Bath
* SOC Media
* Selection plates with desired antibiotic
* Incubator

**Procedure: Time to Transform**

1. Prepare water bath to 42C before starting and leave SOC media out to room temperature.
2. Thaw a tube of NEB 5-alpha Competent E. coli cells on ice for 10 minutes.
3. Add 5 µl containing 1pg - 100ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA
4. Place the mixture on ice for 30 minutes. Do not mix.
5. Heat shock at exactly 42C for exactly 30 seconds. Do not mix.
6. Place on ice for 5 minutes. Do not mix.
7. Pipette 950 µl of room temperature SOC into the mixture.
8. Place at 37°C for 60 minutes. Shake at 300rpm.
9. Warm selection plates to 37°C.
10. Spread 50-100 µl of each dilution onto a selection plate and incubate overnight at 37°C

# *Miniprep*

We utilize the Zymo Low Copy Protocol Miniprep for our experiments.

**Materials**

* Culture of Transformed Cells
* Zymo Miniprep Kit

**Procedure: Plasmid Purification**

1. Centrifuge up to 10 mL of bacterial culture in a clear 1.5 ml tube at full speed for
   15-20 seconds in a microcentrifuge. Discard supernatant.
2. Add 500 ul of cold ZymoPURE™ P1 (Red) to the bacterial cell pellet and
   resuspend completely by vortexing or pipetting
3. Add 500 ul of ZymoPURE™ P2 (Blue) and immediately mix by gently inverting the tube 8-10 times. Do not vortex! Let sit at room temperature for 3 minutes. Cells are completely lysed when the solution appears clear, purple, and viscous.
4. Add 500 ul of ZymoPURE™ P3 (Yellow) and mix thoroughly by inversion. Do not vortex! Invert the tube an additional 5 times after the sample turns completely yellow. The sample will turn yellow when the neutralization is complete, and a yellowish precipitate will form.
5. Centrifuge the neutralized lysate for 5 minutes at 16,000 x g.
6. Transfer exactly 1,200 ul of supernatant from step 5 into a clean 1.5 ml microcentrifuge tube.
7. Add 520 ul of ZymoPURE™ Binding Buffer to the cleared lysate from step 6 and mix thoroughly by vortexing for 15 seconds.
8. Place a Zymo-Spin™ Column in a Collection Tube.
9. Transfer the entire mixture from step 7 into the Zymo-Spin™ Column. Incubate assembly at room temperature for 1 minute and then centrifuge at ≥ 10,000 x g for 1 min. Discard the flow through.
10. Add 800 ul of ZymoPURE™ Wash 1 to the Zymo-Spin™ Column and centrifuge at ≥10,000 x g for 1 min. Discard the flow through.
11. Add 800 ul of ZymoPURE™ Wash 2 to the Zymo-Spin™ Column and centrifuge at ≥ 10,000 x g for 1 min. Discard the flow through.
12. Add 200 ul of ZymoPURE™ Wash 2 to the Zymo-Spin™ Column and centrifuge at ≥ 10,000 x g for 1 min. Discard the flow through.
13. Centrifuge the Zymo-Spin™ Column at ≥ 10,000 x g for 1 minute in order to remove any residual wash buffer.
14. Transfer the Zymo-Spin™ II-PX Column into a clean 1.5 ml tube and add 25 ul of ZymoPURE™ Elution Buffer directly to the column matrix. Incubate at room temperature for 2 minutes, and then centrifuge at ≥ 10,000 x g for 1 minute in a microcentrifuge.
15. Nanodrop and store the eluted plasmid DNA at -20°C.

# *DNA Bead Clean-Up*

**Materials**

* Omega-Biotek Magnetic Bead Based Solution
* Vector and Insert DNA
* 75% Ethanol
* Incubator or Thermocycler

**Procedure: One of Magnetic Powers**

1. Calculate correct volume of bead solution for desired concentration (1x, 0.5x, etc).
2. Thoroughly vortex beads before adding to the tube. Immediately pipette beads and add to the tube directly after vortexing to ensure right concentration!
3. Let beads set on bench for 5 minutes, then place on tray on magnet for 5 minutes.
4. Remove supernatant being careful to not touch the beads attached to the sides of the tube.
5. Repeat the previous step with two washes using 190ul 75% ethanol.
6. Remove all residual ethanol from the tube. Place in incubator or thermocycler with lid open at 37C for ~15 minutes.
7. After beads are fully dry, take tubes off thermocycler and add 18ul of nuclease-free water to each. Gently mix by pipetting and wait for 2 minutes.
8. Place tube with resuspended beads back onto magnet and wait 2 minutes.
9. Supernatant contains the DNA. Carefully place into a clean new tube by pipetting.

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{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
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# Results

# Overview of Experimental Setup

We aimed to utilize isolated enzymes known as laccases as a potential method of combating EE2 pollution of water sources. Laccases are a prominent class of multi-copper oxidases found naturally in numerous species of fungi and bacteria. They are versatile enzymes that are proven to be capable of degrading phenolic compounds and this makes their implementation in industrial and municipal wastewater treatment attractive. We utilized NCBI to find bacterial laccase genes and IDT’s codon optimization tool to ligate the laccase into a vector and transform it into *Esherichia coli*. We initially wanted to use the pET-22b(+) vector (Novagen) because it contains a pelB leader sequence to allow for a system of extracellular secretion. Due to ordering difficulties during the first cycle of the project, we were unable to receive the plasmid but were donated one with an insert (Profilin gene) inserted by XBaI and EcoRI restriction enzymes by Dr.Aoife Heaslip’s lab at UConn. We aimed to remove the insert and replace it with our gblock with compatible restriction enzymes.

![Wastewater](https://static.igem.wiki/teams/4984/wiki/laccase-gene.png)

# Summary of Results

We initially had difficulty digesting the insert and obtaining enough DNA after gel extraction of the digestion. Therefore, we struggled at first to get an initial ligation. Once our pET22b(+)ordered plasmid came in, we attempted to digest it and ligate it again. Since we had difficulty getting good DNA from the gel extractions, we attempted to use magnetic beads to separate the plasmid backbone from the part of the multiple cloning site that was removed. We evaluated inserts of several colonies with primers that annealed to our pET22(+) plasmid, with one colony that had a large enough product to be the gBlock from the *Bacillus licheniformis* laccase. We sent the PCR and minipreps from the plasmid to be sequenced by Eurofins and if successful, we will test if the bacterial strain can biodegrade EE2.

![Wastewater](https://static.igem.wiki/teams/4984/wiki/final-gel-pic.png)
Figure: Colony PCR with pET-22b vector primers

* L: Ladder in first lane
* Lane 8: Sample Colony 7 demonstrates one colony which had the larger product that could be the *B. licheniformis* gene insert into the pET vector.

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