Difference between revisions of "Part:BBa K1819001"
 
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In 2021, team KEYSTONE improved the pre-existing Lcp(LcpK30) by adding new characterization data for it and designing a new Lcp type- Lcp1VH2, Lcp1VH2 has performed better  expression and enzyme activity compared to LcpK30. 
 
In 2021, team KEYSTONE improved the pre-existing Lcp(LcpK30) by adding new characterization data for it and designing a new Lcp type- Lcp1VH2, Lcp1VH2 has performed better  expression and enzyme activity compared to LcpK30. 
  
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<h3>Better expression</h3>
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In order to compare LcpK30 and Lcp1VH2, we tested two proteins using E.coli BL21 with plasmid pET23a:: Lcp1VH2 (C.2 & S.2 presented on figure 1) and pET23a:: LcpK30 (C.3 & S.3 presented on figure 1). From the SDS-Page result in fig.1, Lcp1VH2 expressed a clear and distinct band at 42 kDa, while the band trace of LcpK30 at the same position was obviously much weaker in comparison, indicating that its expression was much inferior to that of Lcp1VH2.
  
 
<div><img src="https://static.igem.org/mediawiki/parts/7/70/T--KEYSTONE--lcp1.png"></img src></div>
 
<div><img src="https://static.igem.org/mediawiki/parts/7/70/T--KEYSTONE--lcp1.png"></img src></div>
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Fig. 1 Expression of Lcp1VH2 and LcpK30 when inserted into pET23a plasmid.SDS-PAGE of crude extracts(C) and soluble fractions(S) of E. coli BL21(1) as control, Lcp1VH2 (2) and fusion proteins LcpK30 (3). 
 
Fig. 1 Expression of Lcp1VH2 and LcpK30 when inserted into pET23a plasmid.SDS-PAGE of crude extracts(C) and soluble fractions(S) of E. coli BL21(1) as control, Lcp1VH2 (2) and fusion proteins LcpK30 (3). 
  
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<h3>Enzyme activity verification</h3>
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<h3>Better enzyme activity</h3>
 
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. The oxygen consumption rate in the sample tube represents the enzyme activity of Lcp1VH2.
 
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. The oxygen consumption rate in the sample tube represents the enzyme activity of Lcp1VH2.
 
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 sample tube containing LcpK30, oxygen was consumed at a slower rate, and after 9.5 hours, oxygen was roughly depleted. However, the dissolved oxygen in the control tube(Supernatant only of BL21) slowly drops to 6mg/l after 24 hours.  
 
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 sample tube containing LcpK30, oxygen was consumed at a slower rate, and after 9.5 hours, oxygen was roughly depleted. However, the dissolved oxygen in the control tube(Supernatant only of BL21) slowly drops to 6mg/l after 24 hours.  
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Fig 2. Dissolved oxygen experiment to verify the activity of Lcp1VH2 and LcpK30 
 
Fig 2. Dissolved oxygen experiment to verify the activity of Lcp1VH2 and LcpK30 
  
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<div>
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<br><br>
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Citations: 
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<br><br>
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1. 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. 
 +
<br><br>
 +
2.    Rose, K., Tenberge, K.B., Steinbüchel, A., 2005. Identification and characterization of
 +
genes from Streptomyces sp. strain K30 responsible for clear zone formation on natural rubber latex and poly(cis-1,4-isoprene) rubber degradation. Biomacromolecules 6, 180–188.
 +
<br><br>
 +
3.      Rose, K., Steinbüchel, A., 2005. Biodegradation of natural rubber and related compounds:
 +
recent insights into a hardly understood catabolic capability of microorganisms. Appl.
 +
Environ. Microbiol. 71, 2803–2812.
 +
 +
 +
</br>
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<h1>Team:SMS_Shenzhen 2021</h1>
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 +
Lcp is widespread in Gram-positive rubber degraders, and is responsible for the primary attack on polyisoprene by actinomycetes.
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Lcp has a molecular mass of about 42 kDa. The amino acid sequences of Lcps have no heme binding motifs that could serve as covalent attachment sites for heme groups.
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 +
<img src="https://static.igem.org/mediawiki/parts/thumb/3/38/T--SMS_Shenzhen--contri3.png/718px-T--SMS_Shenzhen--contri3.png" style="width:400px"><br>
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 +
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Figure.1 Two conformers of Lcp-K30.[1]
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</br>
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LCP is identified as a b-type cytochromes.  Lcp was classed as a globin and the structural importance of several residues contributing to the stability of the protein, especially Arg195 and Arg202 was stated. The conserved residues (Arg164, Thr168 and His198) of the recently biochemically characterised Lcp-specific domain of unknown function 2236 (DUF2236) are located close to the haem cofactor and were identified as crucial active site residues.
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<br>
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<img src="https://static.igem.org/mediawiki/parts/thumb/0/08/T--SMS_Shenzhen--contri4.png/733px-T--SMS_Shenzhen--contri4.png" style="width:400px"><br>
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Figure.2 Degradation products of oxidative cleavage of poly(cis-1,4-isoprene). [2]
 +
</br>
 +
Lcp cleaves rubber to multiple products ranging from C20 tetra-isoprenoid to at least C35 hepta-isoprenoid. The Lcp was assume that is more surface exposed and should be located close to the substrate binding site. Depending on the binding of the polymer to the protein, the substrate can be cleaved at different relative positions with respect to the end of the polymer molecule, resulting in cleavage products that differ largely in the number of isoprene units.
 +
 +
</br>
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</br>
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<h5>Reference: </h5>
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[1]Ilcu, L., Röther, W., Birke, J., Brausemann, A., Einsle, O., & Jendrossek, D. (2017). Structural and Functional Analysis of Latex Clearing Protein (Lcp) Provides Insight into the Enzymatic Cleavage of Rubber. Scientific reports, 7(1), 6179. https://doi.org/10.1038/s41598-017-05268-2
 +
<br>
 +
[2]Birke, J., Röther, W., & Jendrossek, D. (2015). Latex Clearing Protein (Lcp) of Streptomyces sp. Strain K30 Is a b-Type Cytochrome and Differs from Rubber Oxygenase A (RoxA) in Its Biophysical Properties. Applied and environmental microbiology, 81(11), 3793–3799. https://doi.org/10.1128/AEM.00275-15
  
  

Latest revision as of 02:06, 22 October 2021

Latex Clearing Protein (Lcp) - rubber degradation

Latex clearing protein (Lcp) from actinomycete Streptomyces sp. strain K30 (GenBank accession code: AAR25849.1) is a 42kDa enzyme which catalyzes rubber cleavage at the cis double bonds to multiple products ranging from C20 tetra-isoprenoid to at least C35 hepta-isoprenoid with measured specific activity of 1.3 mol/min per mg.

It contains an oxidized heme-Fe3+ not bound to dioxygen.


Figure 1 - Schematic representation of BBa_K1819001 insert

figure 2 - Gel analysis confirmation of Lcp in pSB1C3


We performed a bioinformatics analysis of Lcp predicting its structure, creating an HMM model and comparing RoxA and Lcp secondary structure based on reported data. Click here to see our analysis

Improvement and new characterization data of BBa K1819001 made by Team KEYSTONE 2021

Improvement

In 2021, team KEYSTONE improved the pre-existing Lcp(LcpK30) by adding new characterization data for it and designing a new Lcp type- Lcp1VH2, Lcp1VH2 has performed better  expression and enzyme activity compared to LcpK30. 

Better expression

In order to compare LcpK30 and Lcp1VH2, we tested two proteins using E.coli BL21 with plasmid pET23a:: Lcp1VH2 (C.2 & S.2 presented on figure 1) and pET23a:: LcpK30 (C.3 & S.3 presented on figure 1). From the SDS-Page result in fig.1, Lcp1VH2 expressed a clear and distinct band at 42 kDa, while the band trace of LcpK30 at the same position was obviously much weaker in comparison, indicating that its expression was much inferior to that of Lcp1VH2.
Fig. 1 Expression of Lcp1VH2 and LcpK30 when inserted into pET23a plasmid.SDS-PAGE of crude extracts(C) and soluble fractions(S) of E. coli BL21(1) as control, Lcp1VH2 (2) and fusion proteins LcpK30 (3). 

Better enzyme activity

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. The oxygen consumption rate in the sample tube represents the enzyme activity of Lcp1VH2. 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 sample tube containing LcpK30, oxygen was consumed at a slower rate, and after 9.5 hours, oxygen was roughly depleted. However, the dissolved oxygen in the control tube(Supernatant only of BL21) slowly drops to 6mg/l after 24 hours.   This indicates that the LCP1VH2 protein has a higher enzymatic activity.  
Fig 2. Dissolved oxygen experiment to verify the activity of Lcp1VH2 and LcpK30 


Citations: 

1. 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. 

2. Rose, K., Tenberge, K.B., Steinbüchel, A., 2005. Identification and characterization of genes from Streptomyces sp. strain K30 responsible for clear zone formation on natural rubber latex and poly(cis-1,4-isoprene) rubber degradation. Biomacromolecules 6, 180–188.

3. Rose, K., Steinbüchel, A., 2005. Biodegradation of natural rubber and related compounds: recent insights into a hardly understood catabolic capability of microorganisms. Appl. Environ. Microbiol. 71, 2803–2812.

Team:SMS_Shenzhen 2021

Lcp is widespread in Gram-positive rubber degraders, and is responsible for the primary attack on polyisoprene by actinomycetes. Lcp has a molecular mass of about 42 kDa. The amino acid sequences of Lcps have no heme binding motifs that could serve as covalent attachment sites for heme groups.
Figure.1 Two conformers of Lcp-K30.[1]
LCP is identified as a b-type cytochromes. Lcp was classed as a globin and the structural importance of several residues contributing to the stability of the protein, especially Arg195 and Arg202 was stated. The conserved residues (Arg164, Thr168 and His198) of the recently biochemically characterised Lcp-specific domain of unknown function 2236 (DUF2236) are located close to the haem cofactor and were identified as crucial active site residues.

Figure.2 Degradation products of oxidative cleavage of poly(cis-1,4-isoprene). [2]
Lcp cleaves rubber to multiple products ranging from C20 tetra-isoprenoid to at least C35 hepta-isoprenoid. The Lcp was assume that is more surface exposed and should be located close to the substrate binding site. Depending on the binding of the polymer to the protein, the substrate can be cleaved at different relative positions with respect to the end of the polymer molecule, resulting in cleavage products that differ largely in the number of isoprene units.

Reference:
[1]Ilcu, L., Röther, W., Birke, J., Brausemann, A., Einsle, O., & Jendrossek, D. (2017). Structural and Functional Analysis of Latex Clearing Protein (Lcp) Provides Insight into the Enzymatic Cleavage of Rubber. Scientific reports, 7(1), 6179. https://doi.org/10.1038/s41598-017-05268-2
[2]Birke, J., Röther, W., & Jendrossek, D. (2015). Latex Clearing Protein (Lcp) of Streptomyces sp. Strain K30 Is a b-Type Cytochrome and Differs from Rubber Oxygenase A (RoxA) in Its Biophysical Properties. Applied and environmental microbiology, 81(11), 3793–3799. https://doi.org/10.1128/AEM.00275-15

Please note this part was submitted in pSB1C3, the Registry's standard shipping backbone, according to submission requirements. However, pSB1C3 contains a SacI site and if you want to use this part we recommend to move the part into another plasmid backbone what can be easily done.




Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 889
    Illegal XhoI site found at 1043
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 535
    Illegal NgoMIV site found at 857
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 438