iGEM 2024 — Team bkea

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{% block title %}Lab 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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[●09/09/24](#section1)
[●13/08/24](#section2)
[●17/08/24](#section3)
[●23/09/24](#section4)
[●27/09/24](#section5)
[●30/09/24](#section6)
[●1/10/24](#section7)

### 09/09/24 Goal: Synthesize Calcium Oxalate crystals and prepare LB medium and Agar-plates for further experiments

Participants: Caitlin

* The exact measurements of Na2Ox and CaCl2 was 0,839g and 0,696 respectively
* The pH of the Tris-HCl buffer was 6,46 - it was made form 0,303g TRIS in 200 mL demineralised water. Then the pH was adjusted using 0,1M HCl and 0,1M NaOH before filling up to 250 mL with demineralised water
* There was a crystal formation immediately after adding the two salts, therefore NaCl was not added to the solution
* It sat for 2 weeks to allow the crystals to fall to the bottom and the water to evaporate

### 13/08/24 Goal: Make a serial dilution of hydrogen peroxide to test bacterial survival

Participants: Caitlin, Mads, Anna, Viktor, Jonathan
 Hydrogen peroxide survivability by use of a growth curve

* The experiment went smoothly apart from having to remove 50 µl of water from the control tube to assure that the concentration of bacteria was constant

### 17/08/24 Goal: Look at results from the hydrogen peroxide experiment

Participants: Caitlin, (Mads)

* The plates were removed from the incubator and we took pictures before they were disposed of safely

### 23/09/24 Goal: Transformation of E. Coli

Participants: Caitlin, Mads, Viktor
 E-Coli transformation and Oxalate Oxidase production:
We successfully transformed the E. coli and grew it on petri dishes.

* The CaCl\*6H2O was supposed to be in a solid form, whereas the water had escaped from the powdered crystal formation, so a rather unknown amount CaCl had been added. The added CaCl consisted of around 0,2-0,3 ml of the solution of the separated crystals. We further added sterile water until the volume was 10 ml to dilute the solution. The original solution with the fully crystalized CaCl is unknown.
* The laboratory did not have any supply of gas left for use with a bunsen-burner, so we resorted to burning ethanol instead to keep an open flame above the petri dishes to minimize risk of contamination.

### 27/09/24 Goal: Look at results from transformation and transfer to LB-medium

Participants: Caitlin, Marie, Viktor

* The transformation went perfectly.
* All positive controls had growth and all negative controls had none.

### 30/09/24 Goal: Cell lysis to predate the enzyme tests

Participants: Viktor, Mads

* We successfully isolated the bacteria from the lb medium and used 2 kinds of lysis solution to lyse the cells, so we can later isolate the enzyme.

+ 6 of the Eppendorf tubes with cells were lysed with a bio-rad lysis solution that expired in 2017.
+ One out of the 6 tubes lysed with 10% Tween 20 solution were lost to the overflow beaker.

### 1/10/24 Goal: Prove the presence of the enzyme oxalate oxidase.

Participants: Caitlin, Mads, Viktor, (Karoline)

The goal of today's experiment was to prove the presence of the enzyme oxalate oxidase by demonstrating a reduction in oxalate concentration. We carried out the enzyme activity test by measuring the reduction in oxalate concentration over time, with the aim of confirming the presence and activity of the enzyme produced by the transformed E. coli cells.

* During the experiment, we realized that two of our samples might have been contaminated with protease K, which likely affected the enzyme in those samples, preventing them from working as expected.
* Despite this challenge, we achieved the results we were looking for with the remaining samples. The enzyme successfully degraded oxalate, and the measurements fit perfectly with our hypothesis.
* This was the final day in the lab, and no further actions are planned.

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{% block title %}Engineering Cycle{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect 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.{% endblock %}
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[●The Engineering Cycle](#section1)
[●I Wetlab Engineering Cycle](#section2)
[●In Silico Engineering Cycle](#section3)
[●II Wetlab Engineering Cycle](#section4)
[●Design](#subsection4_1)
[●Build](#subsection4_2)
[●Test](#subsection4_3)
[●Learn](#subsection4_4)

### The Engineering Cycle as a Fluid Model

We started out with a design stage in wetlab engineering cycle, where we proposed a plasmid. At the time we weren't sure we would be able to gain access to a GMO lab, so we jumped into a drylab engineering cycle to produce a software tool that could help us steamline the docking process. With this tool we found problems with our plasmid and therefore started a new wet lab engineering cycle. This is just one of the examples of how wetlab and drylab have helped each other in our project.

![](https://static.igem.wiki/teams/5243/wiki-pages/engineering/full-engineering-cycle.webp)

Figure 1: The engineering cycle used with our project.

### The Ⅰ Engineering Cycle in Wetlab

In the first phase of the IGEM Wetlab engineering cycle, the design stage. We attempted to design a plasmid. We made a general plasmid with the general structures. It was important for us that the RBS had strong affinity to produce many proteins, and that the protein contained a means for protein purification, so that if we ran into problems and wanted to examine the protein, that was a possibility. Having a selection marker was also important as it meant having an automatically integrated test for whether the bacteria had been transformed or not.

![](https://static.igem.wiki/teams/5243/wiki-pages/engineering/plasmid-1.webp)

Figure 2: Our plasmid in the first phase of the engineering cycle.

From there we researched specific parts and built our vector with a vector builder. We found that the pBAD promoter met almost all of our requirements for a plasmid and is generally known as well used and reliable plasmid. The only thing missing was our gene of interest as well as a means for protein purification.

Hence we added our gene of interest, the gene for oxalate oxidase. Upstream of this gene a HIS-tag as well as TEV was added. In this way we could do protein purification with the HIS-tag if we ran into problems and wanted to examine the protein isolated, as well as being able to remove the his-tag with TEV proteases that can recognize the TEV sequence and cut.

![](https://static.igem.wiki/teams/5243/wiki-pages/engineering/plasmid-2.webp)

Figure 3: Our plasmid in the second phase of the engineering cyccle.

To avoid buying all the parts building and testing, and then realizing something was off. We decided to test our gene products with bioinformatic tools. This can be a rather tedious process and with this in mind we started a new engineering cycle, an in silico engineering cycle where we would attempt to simulate the chances of the enzyme encountering natural inhibitors within the body.

### The in Silico Engineering Cycle

In drylab, we used the engineering cycle as a fundamental model to develop the tools that are called LightningAutoDock Tools - in short LAD tools. This was done using this engineering cycle model by first designing and generating the ideas and the functionality that we wanted to implement into our final design to complete the docking tasks. After testing the LightningAutoDock (the python tool running the docking software) and seeing errors such as cmd prompt freezing, instability and slow docking speed, we corrected the script in order to address the issues.

Along with this iterative process there were a lot of debugging, a lot of miscommunication between the AutoDock4 algorithm files and the way it parsed the arguments. This meant that we used the engineering cycle in drylab by first designing, then building, then testing and then learning. After many iterations we had acquired sufficient knowledge of how the AutoDock4 systems worked and then the development of the python tools were easier to complete. This process was done over many weeks in order to finalise the LightningAutoDock Tool suite.

During our docking simulations, we made a significant discovery regarding histidine's ability to bind at the entrance of the active site of our enzyme, oxalate oxidase. The conformation with histidine and the oxalate oxidase had a binding energy of -7.4 kcal/mol. This posed a potential issue, as our purification strategy initially relied on a 6xHis-tag (six histidine residues in sequence). Given the theoretical risk of histidine residues from the tag interfering with enzyme activity, either by blocking the active site or causing aggregation, we decided to replace the His-tag with a GST (Glutathione S-transferase) tag. This switch allowed us to avoid possible complications with enzyme function during purification, even though we were unable to fully implement the GST tag experimentally within our project’s timeframe.

### The II Engineering Cycle in Wetlab

#### Design

With our learned knowledge from our in sillico engineering cycle we once again attempted to make a wet lab engineering cycle and designed a new plasmid. In the in sillico engineering cycle we produced a new software tool and used it to learn that we could not have a HIS-tag on our final product was not viable. So we replaced the HIS-tag with the GST tag, tested the redesigned gene product with the in silico software tool and found no pressing problems. With this in mind we started the building stage.

![](https://static.igem.wiki/teams/5243/wiki-pages/engineering/plasmid-3.webp)

Figure 4: Our final plasmid design

#### Build

We had trouble finding a GMO lab to house our experiments. Therefore, we ordered a pre assembled plasmid form vector builder. For the transformation of the bacteria we used heat shock. and set them to grow our agar plates. With that we had build our “biological machine”.

#### Test

In the testing stage we had set up 2 tests. One general test where we tested that the bacteria had been properly transformed through an antibiotic selection marker.

For the second part of the test stage the bacteria was then transferred into a liquid medium, after having grown in the liquid medium the cells were lysed and centrifuged. In this way we got our enzyme out of the cells and the supernatant could then be transferred to a solution with oxalate to test the effects. We then through titration measured how much we were able to break down.

#### Learn

Our tests showed that we were able to successfully transform our E.Coli. and that the enzymes produced were able to successfully break down oxalate. More information is available on our Results page. In this way we learned that when we transformed E.Coli with this specific plasmid. The E.Coli were able to grow and produce enzymes. When these enzymes were transferred and tested on oxalate, they were able to break down the oxalate. This knowledge gives us something to build upon if we were to go into a new engineering cycle.

Hypothetically if we were to make a new engineering cycle. We might try to redesign our plasmid so it becomes fit for a lactic acid bacteria, and insert it into a lactic acid bacteria capable of yogurt. otherwise we might try to redesign the regulatory part of our plasmid in such a way that oxalate oxidase is expressed only when oxalate is present. In engineering many redesigns and possibilities for optimization lie right in front of us. and it is our choice which ones to choose.

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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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[●The GMO Experiment](#section1)
[●E.coli Resistance](#section2)
[●Results](#section3)
[●Protocols](#section4)

The following are quick descriptions of our experiments, why we made them and their results. To see our more precise lab notes and protocols, please see the Lab Notebook tap above.

### The GMO Experiment - Transformation and Enzyme Test

We wanted to prove that it was possible to transform bacteria with our plasmids, and produce the enzymes that were able to break down oxalate.

To prove it we wanted to create four different agar plates that would contain the following; two with antimicrobial peptides and two without, with one being with the plasmid insert at 2 without.

![](https://static.igem.wiki/teams/5243/wiki-pages/ku-resultater/amp-plasmid.webp)

Our transformation was done with the method of heat shock and we utilized a nickel column to collect the protein of interest. And the enzymes were activated with the use of L-arabinose, where we measured the amount they were able to break down using a titration with potassium permanganate and sulfuric acid

### E.coli Resistance to Hydrogen Peroxide

To make sure we wouldn't end up with zero results, we wanted to test how well E. coli would be able to produce enough Catalase, a hydrogen peroxide degrading enzyme. This was important since the breakdown of our enzymes releases hydrogen peroxide. We knew that bacteria have pathways to break down hydrogen peroxide but we wanted to test how well this feature was, to make sure it was safe. The experiment would hopefully show us whether we needed to add the gene that codes Catalase to our plasmid or if the bacteria were naturally able to survive the increased concentration.

This was simply done by making LB medium containing different levels of hydrogen peroxide, and a control without to determine the survival rate at different levels.

### Results

The results showed that when the concentration of hydrogen peroxide was 0,01% or lower there was no significant decrease in bacterial growth compared to the control. Though at 1% hydrogen peroxide the bacterial growth was severely stunted compared to the control (the solution with bacteria bubbled when the hydrogen peroxide was added showing its oxidative capabilities.) as well as that the 0,1% also had significantly less growth. This shows that concentrations from about 0,1% and above are harmful for the bacteria and therefore most likely also for our cells.

From the experiment we can conclude that at low concentrations of hydrogen peroxide the bacteria are not harmed. Though to go further with the experiment we should test what the concentrations of oxalate are in the patients so that we have an estimate of how much hydrogen peroxide is produced.

### Protocols

* [The GMO experiment - Transformation and enzyme test](https://static.igem.wiki/teams/5243/pdfs/transformation-af-bakterier.pdf)
* [E.coli resistance to hydrogen peroxide](https://static.igem.wiki/teams/5243/pdfs/hydrogenperoxid-vs-overlevelse-via-v-kstkurve-1.pdf)
* [Oxalate Assay](https://static.igem.wiki/teams/5243/pdfs/spectrofotometri-1.pdf)

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