iGEM 2023 — Team wego-taipei

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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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![](https://static.igem.wiki/teams/4748/wiki/loading.gif)

![](https://static.igem.wiki/teams/4748/wiki/notebook.png)

{% extends "layout.html" %}
{% 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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![](https://static.igem.wiki/teams/4748/wiki/loading.gif)

![](https://static.igem.wiki/teams/4748/wiki/engineering-success.png)

[Preparation of DNA Parts](#preparationDNA)

[Type IIS cloning - Level 0](#cloningL0)
[Type IIS cloning - Level 1](#cloning1)
[Evaluating Clone Efficacy and T1D Beta Cell Phenotype Rescue](#evaluatingClone)

Engineering Success

#### Preparation of DNA Parts

After designing our constructs for both the RNLS and Reg3g genes (Figure 1A and 1B), we used Asimov’s Mammalian Collection to acquire the appropriate DNA parts (Table 1), including the EFS promoter, YFP sequences, all five L0 plasmids and all four L1 plasmids.
We performed transformation with DH5⍶ E. coli competent cells to obtain a larger quantity of plasmids for each part. Transformants were screened with ampicillin or spectinomycin, in accordance with information listed on the Airtable.
All of our L0 destination plasmids glow green when subjected to UV light (Figure 2), indicating that the transformation and plasmid extraction processes went smoothly since the superfolder GFP was translated.
We then prepared glycerol stocks of each part with remaining liquid bacterial cultures.
Additionally, given the small size of 1x uORF sequences and 4x uORF sequences, we chose to synthesize these parts to avoid challenges with separating such small fragments through gel electrophoresis.

![](https://static.igem.wiki/teams/4748/wiki/engineering-figure-1a.png)

Figure 1A, Schematics of RNLS

![](https://static.igem.wiki/teams/4748/wiki/engineering-figure-1b.png)

Figure 1B, Schematics of Reg3g

| DNA Parts |
| --- |
| Index | Part Link | Part Type (unified) | Orgin | Collection | Kit Plate | Well | Cutter |
| 219 | BBa\_J433002 | EFS | promoter | BBa\_J433103 | Asimov | Asimov Mammalian Parts Collection | Type IIS L0 Part |
| 201 | BBa\_J433005 | uORFs (1x) | 5' UTR | BBa\_J433104 | Asimov | Asimov Mammalian Parts Collection | Type IIS L0 Part |
| 205 | BBa\_J433006 | uORFs (4x) | 5' UTR | BBa\_J433104 | Asimov | Asimov Mammalian Parts Collection | Type IIS L0 Part |
| 213 | BBa\_J433012 | YFP\_v2\_F\_frag4Overhang | kozak-cds | not added yet | Asimov | Asimov Mammalian Parts Collection | Type IIS L0 Part |
| 213 | BBa\_J433012 | YFP\_v2\_F\_frag5Overhang | kozak-cds | not added yet | Asimov | Asimov Mammalian Parts Collection | Type IIS L0 Part |
| 209 | BBa\_J433045 | L0 promoter | promoter | BBa\_J433103 | Asimov | Asimov Mammalian Parts Collection | Type IIS L0 Part |
| 212 | BBa\_J433045 | L0 5'UTR | 5' UTR | BBa\_J433104 | Asimov | Asimov Mammalian Parts Collection | Type IIS L0 Part |
| 215 | BBa\_J433045 | L0 Kozak-CDS | kozak-cds | BBa\_J433105 | Asimov | Asimov Mammalian Parts Collection | Type IIS L0 Part |
| 217 | BBa\_J433045 | L0 3'UTR | 3' UTR | BBa\_J433106 | Asimov | Asimov Mammalian Parts Collection | Type IIS L0 Part |
| 227 | BBa\_J433045 | L0 polyA | polyA | BBa\_J433107 | Asimov | Asimov Mammalian Parts Collection | Type IIS L0 Part |
| 220 | BBa\_J433046 | L1 TU1 - lacZ | plasmid backbone | BBa\_J433115 | Asimov | Asimov Mammalian Parts Collection | Type IIS L1 Cloning Plasmid |
| 221 | BBa\_J433046 | L1 TU2 - lacZ | plasmid backbone | BBa\_J433116 | Asimov | Asimov Mammalian Parts Collection | Type IIS L1 Cloning Plasmid |
| 222 | BBa\_J433046 | L1 TU3 - lacZ | plasmid backbone | BBa\_J433117 | Asimov | Asimov Mammalian Parts Collection | Type IIS L1 Cloning Plasmid |
| 223 | BBa\_J433046 | L1 TU4 - lacZ | plasmid backbone | BBa\_J433118 | Asimov | Asimov Mammalian Parts Collection | Type IIS L1 Cloning Plasmid |

![](https://static.igem.wiki/teams/4748/wiki/engineering-figure-2.png)

Figure 2. Colonies of transformed L0 plasmids from the Asimov Mammalian Collection grown on LB + ampicillin plates.

![](https://static.igem.wiki/teams/4748/wiki/figure-3-engineering-success.png)

Figure 3, gel map

Coding region sequences of REG3G (CCDS1962.1) and RNLS (CCDS31239.1) were obtained from NCBI. The full length sequence of human REG3G is 536 bp and the full length sequence of human RNLS is 1029 bp. The RNLS ∆ amino oxidase construct consists of the first 300 bp from the 5’ end of RNLS combined with the fragment from 889-1029 bp. Essentially, the amino oxidase domain located between 301-888 bp is truncated in RNLS ∆ amino oxidase . Domain annotations and predictions are obtained from InterPro.

Prior to cloning, we identified a Type IIS cloning illegal site for the BsaI enzyme (5’-GGTCTC-3’) situated at position 796 bp of the human RNLS protein. We made a synonymous substitution to correct the illegal site, thereby preventing the cloning enzymes BbsI and BsaI from cutting our CDS sequence. The sequence of positions 799-801 was changed from TTC to TTT, but both codons code for phenylalanine. To prepare DNA sequences of RNLS and REG3G, we used a human cDNA template from Takara Bio and designed primers containing the required overhang sequences for placing the part into L0 plasmids. These primers include the full length RNLS, RNLS ∆ amino oxidase, and full length REG3G (Table 2).

We used a proofreading polymerase to perform PCR reactions in order to obtain the CDS sequences. Following manufacturer's suggestions, we used an extension time of 30 seconds per kilobase and modified the annealing temperature using online primer calculators and from empirical observations from successive rounds of PCR. Presence and correctness of PCR product was confirmed with gel electrophoresis and 1% or 1.5% TAE gel (Figure 3). Upon visual confirmation with gel electrophoresis, remaining samples are also separated with gel followed by gel extraction and clean up. Once the DNA is purified, DNA concentrations are obtained with Nanodrop and can be used for ligation.

| Name | Sequence | Purpose |
| --- | --- | --- |
| uORF\_4x\_overhang\_included | caacataggttgaaccatgggttaac   ccatgggtgaacatgggttgaacctg   attggttagacaa | Synthesized sequence inclusive of necessary overhangs for Type IIS cloning |
| uORF\_1x\_overhang\_included | caacataggttgaaccatgggttagacaa | Synthesized sequence inclusive of necessary overhangs for Type IIS cloning |
| EFS\_promoter\_F | gctttggcgcctgatgc | Obtain EFS promoter fragment |
| Terminator\_Asimov\_R | tgtaataaaattaaagtagcagtact | Obtain terminator sequence fragment |
| YFP\_v2\_F\_frag4Overhang | cccaGTGAGCAAGGGCGA | Obtain YFP DNA fragment |
| YFP\_v2\_R\_frag4Overhang | actcTTACTTGTACAGCTCGTC | Obtain YFP DNA fragment |
| GaussiaLuc\_F\_frag4Overhang | CCCAGGAGTCAAAGTTCT | Obtain LUC DNA fragmen |
| GaussiaLuc\_F\_frag4Overhang | ACTCTTAGTCACCACCGG | Obtain LUC DNA fragment |
| Reg3g\_cds\_human\_Fnew | GCAAATGCTGCCTCCCAT | Obtain REG3G CDS with PCR |
| Reg3g\_cds\_human\_Rnew | TGGGGGCGTCCTTGAACT | Obtain REG3G CDS with PCR |
| RNLS\_cds\_Nterminus\_F | GCAAATGGCGCAGGTGC | Obtain RNLS CDS with PCR |
| RNLS\_CDS\_Nterminus\_R | TTCTTTCATCACCATTCCTTC | Obtain RNLS CDS with PCR |
| RNLS\_CDS\_Cterminus\_F | CCCAGCTGCCAACTGTCC | Obtain RNLS CDS with PCR |
| RNLS\_CDS\_Cterminus\_R | ACTCGCAATATAATTCTTTAAAGCT | Obtain RNLS CDS with PCR |
| RNLS\_cds\_oxidase\_F | CCCAGCAAGAGTTAGTCTTTCAGCAGCTG | Obtain RNLS CDS with PCR |
| RNLS\_cds1\_R | ACCCACATCCTCAATGCTGTGT | Obtain RNLS CDS with PCR |

#### Type IIS Cloning - Level 0

To make each basic part, each L0 plasmid was digested with the BbsI enzyme to create an opening for ligation of our gene sequences. Digested plasmids are separated through gel electrophoresis and gel extraction and clean up was performed.
Plasmid concentrations are determined with Nanodrop. Ligation was performed using T4 DNA ligase then clones were transformed into DH5α E. coli.
All basic parts are summarized in Table 2 with information detailing which DNA sequence went into each L0 destination plasmid. Original L0 destination plasmids contain a copy of sfGFP, which fluoresces when exposed to UV light.
After digestion and ligation, we expect that these L0 plasmids should contain our genes of interest and not sfGP. Accordingly, our plasmids no longer glow green after ligation.

#### Type IIS Cloning - Level 1

Due to time and lab resource constraints, we were unable to perform our L1 cloning though all L0 parts for our L1 clone are prepared. We have built L0 parts that were destined to make four different kinds of L1 transcriptional units:- 1. EFSpro:REG3G with 4x uORF
- 2. EFSpro:REG3G with 1x uORF
- 3. EFSpro:RNLS WT
- 4. EFSpro:RNLS ∆ amino oxidase.

The two REG3G clones are designed such that we can compare which mode of REG3G overexpression will more positively promote beta cell regeneration when transfected to mammalian cells.
The two uORF sequences should result in different translation efficiencies and REG3G output. Genetic relationships are often complicated and can have different interactions like epistasis, cross-talks, and feedback cycles, meaning that strong overexpression can have unintended negative consequences.
Therefore, we hoped that generating two versions of the REG3G clone can provide preliminary data on translation efficiency and impact on beta cell proliferation. We planned on using this preliminary data to provide empirical input data for further modeling to fine tune REG3G expression and maximize beta cell regeneration.

The two RNLS clones contain a wildtype and one mutant copy with the amino oxidase domain removed. Our main goal for generating these two transcriptional units was to take knowledge of current literature further by evaluating the function of the amino oxidase domain in facilitating beta cells protection in a T1D context.
It was already established in Cai et al. (2020) that mutated RNLS confers beta cell protection, but it remains unclear which parts or domains of the protein are directly involved. We expect that the amino oxidase domain, which should be the RNLS enzyme’s catalytic domain, to be essential in the capability of RNLS to catalyze reactions, and thus focused on this particular domain.
The other domain is the FAD/NAD binding (substrate recognition) domain so we did not focus on this domain.

#### Evaluating Clone Efficacy and T1D Beta Cell Phenotype Rescue

To evaluate the impacts of our clones on T1D, we planned on performing transfection through calcium phosphate transfection. We will also chemically induce T1D in cells using alloxan monohydrate and streptozocin.It is established that both of these drugs can present mammalian cells with symptoms characteristic of T1D (Queiroz et al., 2021).
Following both transfection and induction, we will stain cells with Hoechst and Sytox Orange. Hoechst stains will stain dsDNA and emit blue fluorescence in the process. Sytox Orange will stain dead cells. By calculating the ratio of live to dead cells using both stains, information on the relative impact of each clone on T1D and beta cells can be evaluated.
Additionally, we also planned on extracting total RNA from cell lines to observe how mRNA expression of genes regulating cell division and expansion change using real time quantitative PCR (qPCR).

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{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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![](https://static.igem.wiki/teams/4748/wiki/loading.gif)

![](https://static.igem.wiki/teams/4748/wiki/proof-of-concept.png)

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
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## What should this page contain?

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* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

## Describe what your results mean

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* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

## Project Achievements

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You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

## Inspirations

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* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)

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