iGEM 2022 — Team hk-ssc

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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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## What should this page contain?

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* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

## Inspirations

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* [2018 Munich](http://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

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{% block title %}Engineering Success{% endblock %}
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## Expression of protein mutants

### DESIGN

#### Protein mutants

This year, we wanted to continue optimising the activities of the cyclase for 5AVA, 3-hydroxybutyrate dehydrogenase (CF3HBD). Moreover, we also included lactam-binding NitR as the other potential redesigning target, which takes a critical role in ligand fishing towards the end of our project.

First, we modelled and characterised the ability of several mutants in the dry lab. Mutants were selected based on our calculations of protein stability after iterative energy minimization and simulated point mutations. Structures were further refined by using the Relax and Backrub applications of the Rosetta software suite [[2]](#2). Then, the mutants' activities were compared by QM/MM using the CP2K software package; we selected 8 CF3HBD and 4 NitR mutants to continue testing in the wet lab.

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#### BUILD

The sequences of the CF3HBD mutants are registered as BBa\_K4383004 to BBa\_K4383011, and the sequences of the NitR mutants are registered as BBa\_K4383000 to BBa\_K4383003. Standard cloning techniques were used to insert the CF3HBD and NitR mutants into pET28a and pETBlue2 plasmids respectively. The recombinant vectors, pET28a\_CF3HBD and pETBlue2\_NitR harbouring genes of CF3HBD and NitR respectively, were transformed in *E.coli* DH5α and plated on agar plates with ampicillin for pETBlue2\_NitR and kanamycin for pET28a\_CF3HBD. The transformed clones were screened through colony PCR and the vectors were amplified through plasmid preparation.

For expression of the mutants, the vectors pET28a\_CF3HBD and pETBlue2\_NitR were transformed into *E.coli* NiCo21 cells and plated on agar plates with their respective antibiotics. The LoFT protocol was then used to prepare cell extracts, and the proteins were purified by His-tag purification.

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#### TEST

Due to restraints during the pandemic, we could not analyse the proteins obtained from His-tag purification by SDS-PAGE for their enzymatic properties. We originally planned to analyse protein yields by chemical methods such as IR titration to measure the reaction with substrates.

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#### LEARN

We have learnt how to optimise the factors in the LoFT protocol, such as shaking time, speed, and temperature required to achieve the best yield in our laboratory [[3]](#3).

We plan to finish our final step of confirming protein quality by SDS-PAGE. Our proposed implementation for NitR is to use protein ligand fishing to bind NitR to magnetic beads, and use the protein-bead complex to capture cyclized valerolactam from cell extract.

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## Homologous recombination of *S.elongatus* UTEX 2973

#### Learn and Design

In the first year of our project, we planned using the INTEGRATE System and our shuttle vector panS-loxP-MCS to insert the genes of AK, DavB and DavA into the chromosome of *S.elongatus* UTEX 2973 [[4]](#4). However, in the process of cloning the pSPIN plasmid for the INTEGRATE System, we discovered that cell counts were quite low, likely due to the large 12kb plasmid size putting too much metabolic pressure on the cells. This resulted in low plasmid yields, slowing the entire cloning process, and also indicating that the INTEGRATE System was not the most suitable choice for genomic integration in our project.

Recognizing this difficulty, we changed our method for genomic insertion of *S.elongatus* UTEX 2973. We instead chose the GeneArt™ Synechococcus Protein Expression Vector (pSyn\_6), which allows homologous recombination of exogenous DNA into the natural site 1 of *S.elongatus* [[5]](#5). In our new design we ligate the genes of AK, DavB and DavA from our vector davBA-AK-pUC into pSyn\_6.

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#### Build and test

davBA-AK-pUC was cloned last year through Gibson Assembly. pSyn\_6 and davBA-AK-pUC were transformed into *E.coli* DH5α and plated on agar plates with streptomycin and ampicillin respectively. The vectors were amplified by plasmid preparation.

Due to restraints during the pandemic, we could not carry out restriction enzyme digestion and ligation for inserting the genes of AK, DavB and DavA from our vector davBA-AK-pUC into pSyn\_6. We also could not test electroporation of pSyn\_6 into *S.elongatus* UTEX 2973 to see its resistance to streptomycin. However, we did succeed in transformation by electroporation protocol on *S.elongatus* UTEX 2973 last year.

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#### References

1. Yeom, S.-J.; Kim, M.; Kwon, K. K.; Fu, Y.; Rha, E.; Park, S.-H.; Lee, H.; Kim, H.; Lee, D.-H.; Kim, D.-M.; Lee, S.-G. A Synthetic Microbial Biosensor for High-Throughput Screening of Lactam Biocatalysts. Nature Communications 2018, 9 (1). <https://doi.org/10.1038/s41467-018-07488-0>
2. The Rosetta Software | RosettaCommons <https://www.rosettacommons.org/software>
3. Fujiwara, K.; Doi, N. Biochemical Preparation of Cell Extract for Cell-Free Protein Synthesis without Physical Disruption. PLoS ONE 2016, 11 (4). <https://doi.org/10.1371/journal.pone.0154614>
4. Vo, P. L. H.; Ronda, C.; Klompe, S. E.; Chen, E. E.; Acree, C.; Wang, H. H.; Sternberg, S. H. CRISPR RNA-Guided Integrases for High-Efficiency, Multiplexed Bacterial Genome Engineering. Nature Biotechnology 2020, 39 (4), 480–489. <https://doi.org/10.1038/s41587-020-00745-y>>.
5. GeneArtTM Synechococcus Protein Expression Vector <https://www.thermofisher.com/order/catalog/product/A24230> (accessed 2022 -10 -10).

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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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## What should this page contain?

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Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

## Inspirations

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* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

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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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## 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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