Difference between revisions of "Part:BBa K4015001"

Latest revision as of 22:57, 21 October 2021

Lcp1VH2

The Lcp1VH2 is a strain of latex clearing protein extracted from Gordonia polyisoprenivorans VH2, a species of Gordoniaceae, which is of interest because of its ability to damage natural rubber. Latex clearing protein was first described by Rose (Rose et al., 2005) and the synthesis of Lcp1VH2 into e.coli was first described by Hiessl et. al (Hiessl et al., 2014). The Lcp1VH2 belongs to oxygenate, which can catalyse the extracellular cleavage of poly (cis-1,4-isoprene) through adding two oxygen molecules to the chemical bond where the polyisoprene is attached(Ilcu et al., 2017).

Usage and Biology

In previous studies, we have discovered that lcp1VH2 was usually inserted into the pET23a plasmid and subsequently transferred into the C41 strain for expression. 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 Fig1, 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.  



Fig 1. 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. coli BL21: E. coli BL21(4) as control, pET28a:: Lcp1VH2 (5) and pET23a:: Lcp1VH2 (6).

Improvement

The team Brasil-USP designed the part LcpK30 (Part: BBa_K1819001) in 2015, which is a 42kDa enzyme extracted from actinomycete Streptomyces sp. strain K30 that can catalyze rubber cleavage at the cis double bonds to multiple products ranging from C20 tetra-isoprenoid to at least C35 hepta-isoprenoid.

In this year, we improved the pre-existing Lcp(LcpK30) by adding new characterization data for it and designing a new Lcp type- Lcp1VH2 ,which is a rubber oxygenase extracted by a Gram positive rubber degrading bacteria named Gordonia Polyisoprenivorans VH2.

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 NusA-Lcp1VH2 (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 

Sequence and Features