iGEM 2020 — Team BNDS_China

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# Team:BNDS China/Notebook

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Notebook

NOTEBOOK

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# Team:BNDS China/Engineering

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ENGINEERING SUCCESS

ENGINEERING SUCCESS

Introduction

In our project, we utilized the latest version of SpyTag and SpyCatcher: SpyTag003 and SpyCatcher003 for protein crosslinking1. Particularly, one goal for us to achieve is the gel formation mediated by the interconnection between SpyCatcher003 and SpyTag003. However, to achieve gel formation, the ratio of SpyCatcher003 and SpyTag003 should be optimized. Therefore, from the perspective of engineering, we built our mathematic model to characterize SpyTag003 and SpyCatcher003 by calculating the optimal ratio based on the concept of gelation2.

Modeling Overview

Firstly，we can describe the relationship of gel point Pc and the amount of functionality in SpyTag003
and SpyCatcher003 with the following equation:
In which and are the functionality of SpyTag003 and SpyCatcher003, and are the number of monomers
containing functional groups in SpyTag003 and SpyCatcher003.

![](https://static.igem.org/mediawiki/2020/b/b8/T--BNDS_China--Engineering_Success_Figure_1.png)
*Figure 1. The graph colored as yellow represent C(Na,Nb).*

The graph colored as blue represents the plane whose value is zero.

By Solving for the critical condition where the function equals 0, the two
functions of critical ratio
are attained.
We can therefore derive the function of the maximum and minimum ratio for gelation to occur of the
fraction of Na and Nb：
Where x=Na+Nb，and g1 and g2 are the functions describing the two critical conditions.

By plotting the graph, we find out that both the lower critical bound and upper
critical bound of the
ratio of SpyTag003 and SpyCatcher003 that can form the gel are around 0.8. Therefore, we can conclude
that the best ratio between SpyTag003 and SpyCatcher003 is 0.8.

![](https://static.igem.org/mediawiki/2020/0/0b/T--BNDS_China--Engineering_Success_Figure_2.png)
*Figure 2. The physical map of our hardware — RDR*

See more information in our model webpage.

[Model](https://2020.igem.org/Team%3ABNDS_China/Model)

Reference

1. Keeble, A. H. et al. Approaching infinite affinity through engineering of
peptide-protein interaction. Proc Natl Acad Sci U S A, doi:10.1073/pnas.1909653116 (2019).

2. Flory, P. J. Molecular Size Distribution in Three Dimensional Polymers. I.
Gelation1. Journal of the American Chemical Society 63, 3083-3090, doi:10.1021/ja01856a061 (1941).

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# Team:BNDS China/Experiments

# Experiments

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).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

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# Team:BNDS China/Results

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RESULT

RESULT

Fermentation Of G. Xylinus

We achieved the production of bacterial cellulose through static culture. There is visible bacterial cellulose in the culture flask with HS media
(Figure 1A). Quantitatively speaking, the wet weight of bacterial cellulose produced by G. xylinus in static culture is 17.847g, while the dry weight is
1.012g on average (Figure 1B).

![](https://static.igem.org/mediawiki/2020/9/9a/T--BNDS_China--Result1.png)
![](https://static.igem.org/mediawiki/2020/a/a6/T--BNDS_China--Result2.png)

*Figure 1. Production of bacterial cellulose from static culture.*

(A) The morphology of bacterial cellulose produced by G. xylinus in static culture. (B) Wet weight and dry weight of bacterial cellulose produced in (A). Triplication was
adopted; the error bar illustrates standard deviation.

As for the production of bacterial cellulose by Rotary Disc Reactor (RDR), because of the current pandemic of COVID-19, we haven’t performed
experiments in this part. However, we have successfully constructed the hardware and debugged all the potential errors. We hope to implement
the fermentation via RDR in the next iGEM competition.

Structural Protein Purification

* ![](https://static.igem.org/mediawiki/2020/2/2a/T--BNDS_China--Result3.png)
  *Figure 2. Electrophoresis for the genome PCR.*
* We tried to purify SpyCatcher003 and SpyTag003 based on two
  chassis organisms: and P. Pastoris and E. coli. However, the
  transformation and protein purification process for P. Pastoris are
  both troublesome. For the chemical transformation, the efficiency is
  not so ideal, which means that we need to repeat the
  transformation for many times in order to gain the correct clone. For
  the electroporation, although the efficiency is acceptable, the
  protocol for electroporation equipment is complex and also a little
  dangerous. Therefore, we finally achieved the successful
  transformation of P. Pastoris with genes responsible for
  SpyCatcher003 and SpyTag003 integrated with structural proteins
  according to genome PCR (Figure 2). However, we didn’t finish the
  subsequent protein purification process due to the limit of time.

  Two of the Pichia Pastoris strains GS115 were transformed with genes
  coding SpyCatcher003 with CBM and SpyTag003 with Scl2, respectively,
  under the control of AOX1 promoter. The marker in the electrophoresis
  is composed by DNA bands with length 100bp, 250bp, 500bp, 750bp,
  1000bp, 2000bp, 3000bp, 5000bp, and 8000bp. The lengths of target
  fragments are both about 4000bp, so the electrophoresis report shows
  that transformations are successful.

* ![](https://static.igem.org/mediawiki/2020/4/45/T--BNDS_China--Result4.png)
  *Figure 3. SDS-PAGE result of target proteins.*
* As for the protein purification from E. coli. The SDS-PAGE result
  showed that the proteins is not successfully synthesized. Instead, they
  might be digested to become several fragments of polypeptides with
  different length (Figure 3). The possible reason for the failure of
  protein purification in E. coli might be attributed to the length of
  CDS. The DNA length of SpyCatcher003 with cellulose binding
  module and SpyTag003 with structural proteins are both about 4kb,
  which might be exceedingly long beyond the endurance limit of E.
  coli. Another reason is that protease inhibitor was not adopted in our
  protein purification protocol, which might cause the digestion of our
  target protein.

  Protein bands with different length overlapped with each other to form a
  continuous pattern of the lane as a whole. The pattern suggests digestions
  might occur, cutting target proteins to several fragments of polypeptide with
  different length.

Future Plan

Our project was negatively affected by COVID-19 pandemic. However, our team members for this year and the prospective team leaders for
iGEM 2021 BNDS\_China all feel that this project is worthy to be continued. Therefore, we choose to be a Two-Phase project for the next year
iGEM competition. Our project next year would be based on the theoretical design and modeling prediction done in iGEM 2020, moving forward
to experiment implementation if the pandemic were relieved.

For the production of bacterial cellulose via RDR, we plan to manufacture the hardware in large scale so that we could have multiple equipment
working at the same time to achieve the mass production of bacterial cellulose. Moreover, we decided to adopt Yamanaka Medium Ethanol
(YME), which is a brand-new culture media for the production of bacterial cellulose with more nutrition, in order to enhance the productivity. In
addition, the strain for cellulose production could be various. We plan to make a comparison between G. xylinus, G. hansenii, and several other
kinds of cellulose producing strain in order to find the optimized one.

For the protein purification, we decided to make thorough investigation regarding the fermentation conditions for P. Pastoris. For example, the
concentration of methanol, the inducer for the protein expression in P. Pastoris, is debatable according to academic literature report. Moreover,
the different integration site of linearized expression cassette in P. Pastoris genome would influence the utilization efficiency of methanol,
producing the Mut+ type (normal methanol utilization) and MutS type (slow methanol utilization). The choice of different integration sites is also
debatable, so we decide to try both and adopt the better one. As for the protein expression in E. coli, we plan to verify the endurance limit of this
chassis and add protease inhibitor to avoid digestion of target proteins. If the protein expression in E. coli still didn’t work, we would focus on P.
Pastoris only.

In conclusion, we are looking forward to further continuing this project in iGEM 2021 as a Two-Phase project. With the basis of theoretical design
and analysis, we believe that our experiment implementation would flow well next year.

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