iGEM 2023 — Team unt

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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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## Day-to-Day Lab Details

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### 5/26/23

* Prepared the plasmid DNA retrieved from PCR testing to be sent off to a separate lab for Sanger Sequencing and also introduced that DNA into S17 strain E. coli cells through transformation.

### 5/30/23

* Four plates from transformation failed; redid those 4 plates for transformation. The rest of the plates were successful so we continued onto mating S17 strain E. coli and methanotrophs, using NMS mating media that creates an environment where both can grow with the goal of the target DNA transferring from the E. coli to the methanotrophs. However, since the S17s had little biomass, we restreaked them on new plates for continued growth and for a 2nd mating process. We also made new LB and NMS mating media for bacteria to grow on.

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### 6/6/23

* Met with team colleagues to work on applying for grants and reviewing our research in order to prepare for a presentation with our team manager.

### 6/7/23

* Worked further on presentation topics and on applying for grants with team colleagues.

### 6/8/23

* Same as previous day.

### 6/9/23

* Worked further on presentation, collaborated with team colleagues to prepare to analyze results from methanotrophs, and had meeting with professor on progress made for the week

### 6/12/23

* Met with team to give updates on progress and discussed plans for the week as well as the remainder of the summer. Also analyzed methanotroph cultures after 1 week of growth.

### 6/13/23

* Prepared DH10B strain E. coli cells in liquid culture to be analyzed for fluorescence. Results were inputted in an Excel sheets and graphs were created accordingly.

### 6/14/23

* Preperation of LB Gent/Kan Plates as well as NMS selection and mating media.
* Restreaking of s17 competent cells to prepare for mating.

### 6/15/23

* Mating of s17's with bath.

### 6/16/23

* Restreaked E.coli and methanotroph mating plates on NMS Selection media. Worked with team to finalize responses for grant.

### 6/20/23

* Flourimeter measurement of Anderson Promoter activity in Bath.
* Colony PCR and data entry for the flourimeter results.

### 6/21/23

* Preperation of agragose gel to confirm colony PCR results, DNA cleanup for sequencing, but results were unsatisfactory so we decided not to send off for sequencing.
* Dishes, plastics, other lab upkeep.

### 6/22/23

* Made LB Gent and mating media. Restreaked S17 E. coli on LB Gent media for mating the following day with OB3b strain of methanotrophs.

### 6/23/23

* Mated the S17 E. coli that grew overnight with OB3b methanotrophs and placed the cultures in 30 C incubator to grow over the weekend.

### 7/12/23

* Not applicable labwork.

### 7/13/23

* Not applicable labwork

### 7/17/23

* Second fluorescence measurements in liquid culture bath. Started overnight liquid e. coli cultures.

### 7/18/23

* Fluorescence measurement replicate in e. coli, data entry. Restreaked s17's to prepare for mating with Ob3B. Made Gen10 NMS selection plates for after matings, made mating plates for mating with Ob3B.

### 7/19/23

* What we did here

### 7/20/23 - 9/16/23

* This is the time period where the mutagenesis work was done, mostly by student instructor Spencer Lee.

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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.

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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 %} Some relevant protocols and our rsearch flow.{% endblock %}
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## Our Protocols

#### New England Biolabs Miniprep

We used this miniprep kit in order to obtain appreciable amounts of target DNA for sequencing.

Link

#### New England Biolabs Gibson Assembly

Gibson Assembly was used for the original construction of vectors containing all of the Anderson Promoters.

Link

#### Genemorph II Random Mutagenesis Kit

This protocol was modified slightl to mutate the methane monooxygenase promoter for the creation of the Henard Library.

## Photos from the Lab

### Fluorescence Assay to determine strength of Anderson Promoters in Bath

### Our Biorender Diagram Showing Our Workflow

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{% block title %}Results{% endblock %}
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# Our Project Results

### Please note that due to some of the figures with results being published in a paper in the future all figures will be included in our final presentation for judging but not on the wiki itself.

The relative fluorescence measurements of the Anderson Promoter Series was measured across 3 different microorganisms: Bath, E. coli, and OB3b. These values were obtained by measuring the fluorescence using a fluorimeter and dividing that value by the OD (optical density) to ensure that our data values could be accurately compared. Our data reflected that the promoter strength was strongest in E. coli, followed by Bath, and then OB3b. Specific to promoters, our expectation was for p100 to have the strongest fluorescence, given that it was strongest in the original Anderson Series measurements. However, for our experiment, p104 demonstrated the strongest fluorescence. This was excluding p119, the control promoter that all our promoters were based off of, understandably having the greatest fluorescence. We further compared the correlation of relative fluorescence activity between E. coli and Bath, and E. coli and OB3b in separate graphs. In our graphs, each promoter was displayed to have little variation in fluorescence activity across all microorganisms. This was seen through our lines of best fit having R2 values of 0.854 and 0.742 in graphs comparing OB3b and E. coli and Bath and E. coli, respectively. However, a gap was clearly present in our fluorescence activity between our strongest promoter (p104) and the control (p119). When scaled, our fluorescence activity between the two promoters was too large for our purposes. We were missing activity in the same ranges that would have allowed for successful expression of synthetic products without being high enough to restrict cellular growth.
Our solution involved the creation of a novel mutagenesis promoter library. Twenty five colonies were eventually selected within our desired range of fluorescence. Our data indicated that we had a consistent range of activity in a similar scaled pattern as in previous graphs and sectioned off in ranges from low, medium, to high. This data was precisely what was able to ‘fill the gap’ that we had been challenged with. Further sequencing of these promoters displayed that many of these promoters had similar mutations, and thus five unique promoters were further selected with strengths that were relevant for our interests. When comparing the strengths of our mutated promoters to the original scale that the Anderson Series was measured on, a scale normalized to p100, we saw that the mutated promoters generally had much greater activity, both in E. coli and Bath. The comparison of relative strengths between Bath and E. coli further demonstrated the mutated promoters greater levels of fluorescence in Bath as well. Overall, our results indicate that these mutagenized promoters allow for a full dynamic range of expression without inhibiting cellular growth in both E. coli and Bath. For our purposes, this meant that these promoters would serve as more diverse, flexible, and effective genetic tools, with broad implications on the environment and industrial fields.

Our future plans for the project involve the application of our products to produce viable products. The mutagenized promoters have the ability to convert methane into methanol, serving as a foundation for further conversions into other useful products. Furthermore, these promoters can be further tested in other organisms, and observed to determine whether their efficacy remains the same or if any significant differences occur. OB3b remained out of our mutagenized data due to time constraints and the nature of its slow growth. Further work can be done to see whether OB3b provides similar data as in E. coli and Bath.
Regarding the replication of our experiment, our data has already been repeated in numerous trials to result in the averaged values that are seen in our data. Measures, such as the evaluation of fluorescence in both liquid and cell cultures, as well as in biological and technical replicates, have ensured that our results are precise and accurate and take into account various scenarios. Furthermore, outliers in the general data set have been included in our data to maintain full transparency and indicate that despite those differences, our data as a whole remains reliable.

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