iGEM 2022 — Team bioplus-china

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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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Notebook

[NOTEBOOK1](https://static.igem.wiki/teams/4413/wiki/experiment/notebook1.pdf)
[NOTEBOOK2](https://static.igem.wiki/teams/4413/wiki/experiment/note-book-2.pdf)
[NOTEBOOK3](https://static.igem.wiki/teams/4413/wiki/experiment/notbook3.pdf)

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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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# Engineering Success

## Cycle 1

#### Design

The primary structure of PySp1 (R in brief) includes repeated sequences and N-terminal and C-terminal. The function of N-terminal and C-terminal
is to promote the assembly of spider silk protein to form spider silk fiber and the function of the repeat region is mainly related to the
mechanical properties of spider silk fiber.

We planned to design a complete pathway for expression of 1- 3 repeats in Escherichia coli, and therefore, we first constructed an expression
plasmid containing a repeat sequence, and expressed it in E.coli (BL21 strain), and then explored its physicochemical characteristics through subsequent characterization experiments.

#### Build

We obtained the repeat region sequence of protein PySp1 from the literature, and named it R in brief, and designed a prokaryotic expression
system to express it. The composition part consist of T7 Promoter (BBa\_I719005)、RBS (BBa\_B0034)、R(BBa\_K4413002)、T7 Terminator(BBa\_K731721)
(Figure 1). The T7 promoter controls the start of transcription, RBS sequence combines with ribosome to initiate the protein expression, and the
6X His sequence were inserted before the R for further protein purification. The target fragment was synthesized by a biological company, and then
we recombined the R gene into plasmid with BamHⅠ and EcoRⅠ (Figure 2). In the end, we have successfully got the pET21a-R plasmid (Figure 3),
and transformed it into E.coli (BL21 strain).

![](http://parts.igem.org/wiki/images/4/40/R1_gene_path.png)

Figuer 1: Constitution of T7 Promoter-RBS-R-T7 Terminator gene circuits.

![](http://parts.igem.org/wiki/images/7/7a/R2_Ligation_reaction.png)

Figuer 2: Ligation reaction of pET21a-R with T4 DNA ligase

![](http://parts.igem.org/wiki/images/5/5d/R3_plasmid_map.png)

Figuer 3: Recombinant Plasmid Map of pET21a-R
pET21a vector contains Amp resistant fragments for screening positive clones

#### Test

We verified the length of R and DNA sequence through PCR (Figure 4) and sequence alignment (Figure 5), The following results can prove that we
have successfully constructed the prokaryotic expression system of R.

![](http://parts.igem.org/wiki/images/c/cf/R4_PCR.png)

Figuer 4: Electrophoresis of PCR products amplified from pET21a-R plasmid.
M: DNA Marker；1 and 2：PCR results of positive clone; Product size: 906bp.

![](http://parts.igem.org/wiki/images/8/87/R5a_peak_map.png)
![](http://parts.igem.org/wiki/images/4/47/R5b_Sequence_alignments.png)

Figure 5：The results of Sequencing and Sequence alignments with expected sequence.
a: Sequencing peak diagram;
b: Sequence alignment between R and positive clone sequencing.

![](http://parts.igem.org/wiki/images/5/5d/R3_plasmid_map.png)

Figuer 6: Recombinant Plasmid Map of pET21a-R
pET21a vector contains Amp resistant fragments for screening positive clones

The R expression in E. coli was induced, and the bacterial solution was preliminarily verified by SDS-PAGE (Figure 6). After that,
the target bands in the gel were cut off for mass spectrometry analysis (Figure 7).

![](http://parts.igem.org/wiki/images/b/ba/R7_protein_peak_map.png)

![](http://parts.igem.org/wiki/images/e/ea/R7b.png)

Figure 7: The Mass Spectrometry of expressed Prorein R

The LC-MS analysis indicates that 13 unique peptides cover >50% amino acid sequence of Protein R can be detected by Thermo Q-exactive MS.
This result is also further proof that the target band is indeed the Protein R. Finally, we purified the Protein R from the inclusion body with urea.

#### Learn

After extensive experimental validation, we constructed recombinant plasmids and expressed them in E.coli.
Finally we purified the Protein R from the inclusion body with urea, and mixed them with sodium alginate solution to form a composite hydrogel
as a bioink feedstock for subsequent 3D printing. At the same time, in the Human Practice part, we realized the importance of intuitively observing
the distribution of Protein R in hydrogels. Furthermore, both the tracking and maintenance of the bioink need a reliable indicator. Therefore,
we decided to insert a fluorescent tag before the R gene to tracing the Protein R in our system.

## Cycle 2

#### Design

BBa\_K4413008 contains RBS and R genes. We plan to improve this cassette, and constructed the improve part (BBa\_K4413009). We inserted an EGFP
sequence in the upstream of R. The composition part consist of pT7(BBa\_I719005), RBS(BBa\_B0034)、EGFP(BBa\_S03452), and R（BBa\_K4413002）（Figure 1)。

![](http://parts.igem.org/wiki/images/7/73/R%2BEGFP_1.png)

Figure 1： Constitution of T7 Promoter-RBS-EGFP-R-T7 Terminator gene circuits.

#### Build

We designed a prokaryotic expression system with fluorescent tags, inserting the EGFP sequence upstream of the R to make the protein show green
fluorescence. The EGFP fragment is cloned from the plasmid by PCR, and the 5'end of the primer used in PCR contain the upstream and downstream
homology arms of the pET21a gene respectively，the PCR fragment size is 1395 bp (Figure 2). Integration of EGFP fragments and linearized vectors
pET21a through homologous recombination (Figure 3), Finally, we successfully recombined the pET21a-EGFP-R plasmid (Figure 4), and transferred it
into E. coli (BL21) strain.

![](http://parts.igem.org/wiki/images/b/b9/R%2BEGFP_2.png)

Figure 2：Electrophoresis of PCR products amplified from plasmid.
M: Marker; 1、2: EGFP（720bp）

![](http://parts.igem.org/wiki/images/c/c9/R%2BEGFP_3.png)

Figure 3：Homological recombination of pET21a-EGFP-R
Vector：Linearized vector pET21a-R；Fragment：EGFP

![](http://parts.igem.org/wiki/images/f/f7/R%2BEGFP_4.png)

Figure 4：Recombinant Plasmid Map of pET21a-EGFP-R
pET21a vector contains Amp resistant fragments for screening positive clones

We verified the length of EGFP-R sequence by PCR (Figure 5), and induced the BL21 strain by IPTG , Then we can observed obvious green
fluorescence with fluorescence microscope.

![](http://parts.igem.org/wiki/images/f/f7/R%2BEGFP_5.png)

Figure 5：Electrophoresis of PCR products amplified from pET21a-EGFP-R plasmid.
M: Marker; 1、2: PCR results of positive clone; Product size (EGFP-R): 1600bp

#### Test

EGFP is an excellent reporter gene, which can mark and locate the fusion protein. Through fluorescence microscope, we can accurately locate
the expression position of protein R (Figure 6), and we mix the purified protein with sodium alginate hydrogel (Figure 7), making our product
more intuitive and convenient for positioning in subsequent experiments.

![](http://parts.igem.org/wiki/images/d/d7/R%2BEGFP_6.png)

Figure 6：E.coli cells under fluorescence microscope
a：E.coli with R；b：E.coli with EGFP-R

![](http://parts.igem.org/wiki/images/9/9e/R%2BEGFP_7.png)

Figure 7：Hydrogel with EGFP-R and normal R under blue light
a:Mixed hydrogel with Sodium alginate and Protein EGFP-Rbr
b:Mixed hydrogel with Sodium alginate and Protein R

#### Learn

We have successfully constructed the prokaryotic expression vector of EGFP-R fusion protein. Compared with the original part, the fusion
protein can be directly observed by fluorescence microscope or even eyes. Thus, we can now both increase the mechanic properties of the hydrogel,
and locate our printed biomaterials for further tracing and maintenance.

#### Reference

Blasingame E, Tuton-Blasingame T, Larkin L, Falick AM, Zhao L, Fong J, Vaidyanathan V, Visperas A, Geurts P, Hu X, La Mattina C, Vierra C.
Pyriform spidroin 1, a novel member of the silk gene family that anchors dragline silk fibers in attachment discs of the black widow spider,
Latrodectus hesperus. J Biol Chem. 2009 Oct 16;284(42):29097-108. doi: 10.1074/jbc.M109.021378. Epub 2009 Aug 7. PMID: 19666476;
PMCID: PMC2781455.

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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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## What should this page contain?

---

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

---

* [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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{% 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

---

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

---

* [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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