iGEM 2021 — Team KEYSTONE

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# Team:KEYSTONE/Notebook

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![](https://static.igem.org/mediawiki/2021/3/3c/T--KEYSTONE--project_lab_notes.png)

* [Our clients](#1)
  + [Medical workers](#2)
  + [Practitioners and local residents who handle rubber](#3)
* [Real world application ——Industrial fermentation](#4)
* [Safety considerations](#5)

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# Team:KEYSTONE/Engineering

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# Team:KEYSTONE/Experiments

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# 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

* [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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# Team:KEYSTONE/Results

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![](https://static.igem.org/mediawiki/2021/2/2e/T--KEYSTONE--result_longshot.jpg)

* [Overview](#1)
* [Optimization of Lcp expression system](#2)
* [An efficient Lcp——Lcp1VH2](#3)
* [More Lcp1VH2——the Influences of NusA](#4)
* [HylA – the most efficient signal peptide](#5)
* [Lcp1VH2 Secretion](#6)

Overview

This year we expressed a new latex clearing protein (Lcp1VH2) and enhanced its yield and solubility by NusA and made it autocrine by laccase and HlyA to achieve a more convenient and sustainable process of Lcp degradation of rubber.This webpage is about the five parts of our experiment, which are:

1. Optimization of Lcp expression system:

From the essay, we learned that Lcp1VH2 ——a common Lcp for heterologous expression,is usually inserted into the pET23a plasmid and subsequently transferred into the C41 strain. However, our result showed that this is not the optimal expression system.We inserted Lcp1VH2 into pET23a and pET28a plasmids and then transferred it into the *E. coli* strains BL21(DE3) and C41(DE3), respectively, to explore the optimal expression system among the four combinations. The results are shown in the following figure.

2. An efficient Lcp——Lcp1VH2:

According to the past iGem parts uploaded, we found out LcpK30 is the most common used one in all different teams. However, we made a deeper study, we found Lcp1VH2 may be more effective.We tested the expression and enzyme activity by SDS-PAGE and oxygen consumption experiment respectively.we found Lcp1VH2 has a higher yield and a better enzyme activity when expressed heterologously .

3.More Lcp1VH2——the effects of adding NusA:

Based on previous articles, we found that Lcp1VH2 has a low yield which is led by its low solubility. NusA can work as a solubility enhancer when it is connected with Lcp1VH2.It is known from the article the solubility of the recombinant protein (NusA-Lcp1VH2) is substantially enhanced compared to the poor solubility of lcp1VH2. However, our results show that the yield of the recombinant protein (NusA-Lcp) itself is enhanced even more compared to solubility.

4. The selection of signal peptide:

To facilitate real-time rubber degradation replacing traditional protein utilization processes ,we selected three signal peptides HlyA, Gene III and DsbA and used p47 vector (with GFP) to express these three signal peptides. The p47-GFP-Hlya bacteriophage showed the lowest fluorescence intensity, implying the strongest secretion ability, with a large amount of protein being dispersed into the culture medium.

5. The autocrine of Lcp:

We chose laccase as a more obvious indicator of Lcp1VH2 autocrination. The color reaction called bullish-green occurs when laccase comes in contact with ABTS in the medium. unexpectedly we found that laccase itself also has a secretory effect.Eventually, we add laccase and hlya to both sides of Lcp1VH2 to secrete.The secreted protein has enzymatic activity for rubber degradation

Optimization of Lcp expression system

The latex clearing protein (Lcp) in general is heme b dependent. Its molecular weight is approximately 40kDa. Lcp has a sequence length of 407AA and is used to break down the bonds in between polyisoprene, thus reaching the goal of rubber biodegradation.
In previous studies, we have discovered that lcp1VH2 ——a common Lcp for heterologous expression in *E.coli*，was usually inserted into the pET23a plasmid and subsequently transferred into the C41 strain for expression(Fig1).

![](https://static.igem.org/mediawiki/2021/0/07/T--Keystone--safety_optimization.png)

![](https://static.igem.org/mediawiki/2021/a/a9/T--Keystone--results_opt_description1.png)
We tried that approach, but the bands of Lcp1VH2 were indistinguishable and could not tell whether it was expressed in *E. coli* C41.In order to obtain a viable expression system, we tested four expression protocols using existing strains on hand (*E.coli* C41 and *E.coli* BL21) with vectors (pet23a and pet28a): *E.coli* C41 pET23a:: Lcp1VH2, *E.coli* C41 pET28a:: Lcp1VH2, *E. coli* BL21 pET23a:: Lcp1VH2, *E. coli* BL21 pET28a:: Lcp1VH2 and *E. coli* BL21 pET28a:: cp1VH. coli BL21 pET23a:: Lcp1VH2 and *E. coli* BL21 pET28a:: Lcp1VH2. as shown in Figure 2,we can see that *E. coli* BL21 pET23a:: Lcp1VH2 is the best system for Lcp1VH2 expression: compared to the control, we can see a clear induction band and the Lcp1VH2 expression and water solubility were excellent.
![](https://static.igem.org/mediawiki/2021/f/f9/T--Keystone--results_opt2.png)

Fig 2. Optimization of the expression system of Lcp1VH2. SDS-PAGE of crude extracts(C) and soluble fractions(S) . A. Expression of pET23a::Lcp1VH2 and pET28a::Lcp1VH2 in *E. coli* C41: *E. coli* C41(1) as control, pET23a:: Lcp1VH2 (2)and pET28a:: Lcp1VH2 (3). B. pET23a:: Lcp1VH2 and expression of pET28a:: Lcp1VH2 in E. *E. coli* BL21: *E. coli* BL21(4) as control, pET28a:: Lcp1VH2 (5) and pET23a:: Lcp1VH2 (6).

An efficient Lcp——Lcp1VH2

We found a kind of Lcp on the website of iGEM Parts called LcpK30（BBa\_K1819001）.LcpK30 is from streptomyces sp. K30, a b-type cytochrome, acts as an endo-type dioxygenase producing C20 and higher oligo-isoprenoids. These productions differ in number of isoprene units, while having the same terminal functions, CHO-CH2-, and -CH2-COCH3.

However, since we made a deeper study, we found the existence of another Lcp(Lcp1VH2), and even the new Lcp may be more effective than LcpK30. Lcp1VH2is from Gordonia polyisoprenivorans VH2. It is extremely active in citro degradation, and Lcp1VH2 is produced heterologously with a high yield.

We tested the expression of the two types of Lcp in *E.coli* BL21 after inserting pet23a plasmid to select the lcp we used in the project. Based on the SDS-PAGE, we have found that Lcp1VH2 has a better expression in *E.coli* BL21. On the other hand, the stripe for LcpK30 remains unidentifiable.

![](https://static.igem.org/mediawiki/2021/6/65/T--Keystone--results_lcp1.png)

Fig 3. The comparison of expression between LcpK30 and Lcp1VH2. SDS-PAGE of crude extracts(C) and soluble fractions(S). BL21 as control (1), Lcp1VH2 (2), and LcpK30 (3).

The process for Lcp to degrade rubber requires oxygen consumption. It utilizes the process of β oxidation to break down bonds within polyisoprene. During β oxidation, Lcp adds two oxygen molecules in between the chemical bonds of polyisoprene. Therefore, we have conducted an oxygen consumption test to determine the enzyme activity of these two Lcps to select the better one.

As shown from the oxygen dissolving results below, the initial dissolved oxygen in the sample is about 8.5 mg/l. After 6 hours, the dissolved oxygen in the sample tube (Supernatant containing Lcp1VH2) creates a downward slope. The value dropped to approximately 0mg/l eventually. In the tube containing LcpK30, the speed of oxygen consumption is relatively slower. The oxygen was completely consumed after 9.5 hours.However, the dissolved oxygen in the control tube (Supernatant only of BL21) slowly drops to 6mg/l after 24 hours. This proves that protein Lcp1VH2 has a higher enzyme activity.

![](https://static.igem.org/mediawiki/2021/b/bb/T--Keystone--Lcp2.png)

Fig 4. Oxygen consumption experiment to verify the activity of Lcp1VH2 and LcpK30

Comparing to LcpK30, Lcp1VH2 has a higher protein expression and a higher enzyme activity . Therefore, we selected Lcp1VH2 as the Lcp that is applied within our project.

More Lcp1VH2——the Influences of NusA

The solubility of Lcp1VH2 played an important role in the production yields. Based on previous articles, we found that the solubility of Lcp1VH2 is extremely low. The article brought up a solution, which claims that putting a highly soluble protein to a protein with a low solubility can facilitate protein synthesis. It states that fusing the targeted enzyme to a highly soluble protein can improve the yield of recombinant protein, prevent proteolysis, and increase its solubility. Furthermore, the research paper also suggests that N-utilizing substance A (NusA) is effective at enhancing the synthesis of Lcp1VH2.

![](https://static.igem.org/mediawiki/2021/c/ca/T--Keystone--results_nusa_1.png)

Fig 5. the benefits of NusA demonstrated by the essay

Therefore, we have inserted NusA as the fusion partner to Lcp, creating a new sequence: NusA-Lcp. After comparing this new sequence NusA-Lcp with the original Lcp, we have discovered that even though the water solubility of the new sequence is indeed higher, the water solubility of Lcp is not as low as the research paper has highlighted. (Figure 6)

Fortunately,NusA showed unexpected advantages in our project. Comparing to Lcp1VH2,We have observed an increase on yield of composite protein led by NusA(Fig.6),which is not inconsistent with the enhancement of solubility .The productivity of Lcp1VH2 benefits the later progression of our project.

![](https://static.igem.org/mediawiki/2021/2/20/T--Keystone--NusA_2.png)

Fig 6. NusA’s influence as a fusion partner. SDS-PAGE of crude extracts(C) and soluble fractions(S) of *E. coli* BL21(1) as control, Lcp1VH2 (2) and fusion proteins NusA-Lcp1VH2 (3).

Based on result above, the yield of fusion protein NusA-Lcp increased. unfortunately, the enzyme activity rate of Nus-Lcp1VH2 did not increase comparing to Lcp1VH2, and even decreased. Compared to the sample tube(Supernatant containing Lcp1VH2)，the time for dissolved oxygen dropping to approximately 0mg/l increased from 6 hours to about 16.5 hours in another sample tube(Supernatant containing NusA-Lcp1VH2). This suggests the increase in yield by fusion protein does not increase the enzyme activity.

![](https://static.igem.org/mediawiki/2021/3/3b/T--Keystone--NusA_3.png)

Fig 7. Oxygen consumption experiment to verify the activity of Lcp1VH2 and NusA-Lcp1VH2

HylA – the most efficient signal peptide

Nature has a considerable collection of signal peptides, which are usually constituents of enzyme terminus. First, we identified the type of secretion system suitable for this project: type I secretion system, a one-step extracellular secretion pathway. Three signal peptides are found to be effective and comparable: HlyA, GeneIII, and DsbA.

![](https://static.igem.org/mediawiki/2021/1/14/T--KEYSTONE--project_results_tags.png)

Fig 8. Bacterial secretion systems

![](https://static.igem.org/mediawiki/2021/4/45/T--Keystone--results_HlyA_chart.png)

We used p47 vector containing GFP to compare the effectiveness of these signal peptides. p47-GFP-HlyA displayed a lower level of fluorescence comparing to p47-GeneIII-GFP, suggesting HlyA is more effective in secretion since more GFP has entered the medium. p47-DsbA-GFP showed no fluorescence, meaning protein expression was inhibited or the folding of the protein has failed.
![](https://static.igem.org/mediawiki/2021/c/cc/T--Keystone--results_HlyA_2.png)

Fig 9. Result of GFP secretion experiment

Lcp1VH2 Secretion

To prove that secretion took place, we used laccase as the reporter instead of GFP because GFP does not provide a definite result of whether Lcp is secreted or not. This design is based on the impermeability of the bacterial cell wall to the substrate of laccase, ABTS, so laccase will only react with ABTS and convert it into colorful oxidized ABTS when it is secreted. And since we dissolved ABDS in LB agar, secretion of Lcp will become visible since it is connected to laccase via a linker. We created p47-laccase-Lcp, p47-laccase-Lcp-HlyA, p47-laccase-HlyA, and pET28-Lcp-HlyA as the control to test our hypothesis. In the results, the control group (Fig.10A), as expected, showed no sign of coloration due to the absence of laccase; in the sample containing p47-laccase-Lcp (Fig.10B) secretion happened which implies that laccase by itself can act as a secretion agent; for p47-laccase-HlyA, no secretion happened, and we suspect that HlyA and laccase might suppress each other’s activity(Fig.10C); however, when laccase and HlyA are positioned at the two ends of Lcp, this conflict is resolved(Fig.10).

![](https://static.igem.org/mediawiki/2021/2/2e/T--Keystone--Lcpsecretion_2.png)

Fig 10. A. Strain containing p28a-lcp-hlyA; no coloration observed. B. Srtain containing p47-laccase-lcp; coloration was observed, indicating success in secretion. C. Strain containing p47-laccase-hlyA; no coloration observed. D. Strain containing p47-laccase-lcp-hlyA; coloration was observed, indicating success in secretion.

To examine the signal peptide’s capacity in secreting Lcp, we designed the plasmid pET23a-laccase-Lcp1VH2-HlyA and expressed in *E.coli* BL21.The expression of target protein after iptg induction showed a clear band in Fig.11. To test the activity of Lcp1VH2 secreted autonomously for rubber degradation, cis-1,4polyisoprene granules are mixed with culture fluid and the supernatant in a centrifuge tube respectively, and a change in dissolved oxygen content by time was measured by a dissolved oxygen probe.The culture fluid is the metabolites of *E. coli* BL21 pET23a::laccase-Lcp1VH2-hlya(including the secreted Lcp1VH2), the supernatant is the soluble protein after lysis and centrifugation. Results in Fig.12 shows that Lcp1VH2 in both supernatant and culture medium displayed oxygenase activity, and the concentration of Lcp1VH2 appears to be higher in the supernatant than in the culture medium.

![](https://static.igem.org/mediawiki/2021/f/fe/T--KEYSTONE--project_results_LCsecreting.png)

Fig 11. A.SDS-PAGE of crude extracts(C) and soluble fractions(S) of *E. coli* BL21(1) as control, Lcp1VH2 (2) and fusion proteins laccase-Lcp1VH2-hlya (3). B.Oxygen consumption reaction using supernatant and culture fluid of *E.coli* BL21 pET23a::laccase-Lcp1VH2-HlyA.

Reference

Altenhoff, A. L., Thierbach, S., & Steinbüchel, A. (2020). High yield production
of the latex clearing protein from Gordonia polyisoprenivorans VH2 in fed
batch fermentations using a recombinant strain of Escherichia coli. Journal of
biotechnology, 309, 92–99.

Altenhoff, A. L., Thierbach, S., & Steinbüchel, A. (2021). In vitro studies on the degradation of common rubber waste material with the latex clearing protein (Lcp1VH2) of Gordonia polyisoprenivorans VH2. Biodegradation, 32(2), 113–125.

Andler, R., Heger, F., Andreeßen, C., & Steinbüchel, A. (2019). Enhancing the synthesis of latex clearing protein by different cultivation strategies. Journal of biotechnology, 297, 32–40. https://doi.org/10.1016/j.jbiotec.2019.03.019

Blaudeck, N., Sprenger, G. A., Freudl, R., & Wiegert, T. (2001). Specificity of signal peptide recognition in TAT-dependent bacterial protein translocation. Journal of Bacteriology, 183(2), 604–610.

Burdette, L. A., Leach, S. A., Wong, H. T., & Tullman-Ercek, D. (2018). Developing gram-negative bacteria for the secretion of heterologous proteins. Microbial Cell Factories, 17(1).

Chen, H., Kim, J., & Kendall, D. A. (1996). Competition between functional signal peptides demonstrates variation in affinity for the secretion pathway. Journal of Bacteriology, 178(23), 6658–6664.

Cristobal, S. (1999). Competition between sec- and TAT-dependent protein translocation in escherichia coli. The EMBO Journal, 18(11), 2982–2990.

Dastjerdeh, M. S., Marashiyan, M., Boroujeni, M. B., Golkar, M., Shokrgozar, M. A., & Rahimi, H. (2019).

In silico analysis of different signal peptides for the secretory production of recombinant human keratinocyte growth factor in escherichia coli. Computational Biology and Chemistry, 80, 225–233.

D. C. Thompson, O. Tantot, H. Jallageas, G. E. Ponchak, M. M. Tentzeris and J. Papapolymerou, "Characterization of liquid crystal polymer (LCP) material and transmission lines on LCP substrates from 30 to 110 GHz," in IEEE Transactions on Microwave Theory and Techniques, vol. 52, no. 4, pp. 1343-1352, April 2004.

Freudl, R. (2018). Signal peptides for recombinant protein secretion in bacterial expression systems. Microbial Cell Factories, 17(1).

Hiessl, S., Böse, D., Oetermann, S., Eggers, J., Pietruszka, J., & Steinbüchel, A. (2014). Latex clearing protein-an oxygenase cleaving poly(cis-1,4-isoprene) rubber at the cis double bonds. Applied and environmental microbiology, 80(17), 5231–5240.

Janusz, G., Pawlik, A., Świderska-Burek, U., Polak, J., Sulej, J., Jarosz-Wilkołazka, A., & Paszczyński, A. (2020). Laccase properties, physiological functions, and evolution. International Journal of Molecular Sciences, 21(3), 966.

Kim, S.-K., Park, Y.-C., Lee, H. H., Jeon, S. T., Min, W.-K., & Seo, J.-H. (2014). Simple amino acid tags improve both expression and secretion ofcandida antarcticalipase B in Recombinantescherichia Coli. Biotechnology and Bioengineering, 112(2), 346–355.

Korotkov, K. V., & Sandkvist, M. (2019). Architecture, function, and substrates of the type II secretion system. Protein Secretion in Bacteria, 227–244.

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