Difference between revisions of "Part:BBa K1819001"

Revision as of 15:28, 20 October 2021 (view source) Anrdew (Talk | contribs) ← Older edit		Revision as of 15:31, 20 October 2021 (view source) Anrdew (Talk | contribs) Newer edit →
Line 34:		Line 34:
	This indicates that the LCP1VH2 protein has a higher enzymatic activity.		This indicates that the LCP1VH2 protein has a higher enzymatic activity.
−	<img src="https://2021.igem.org/wiki/images/6/68/T--KEYSTONE--Lcp2.png"></img src>	+	<div><img src="https://2021.igem.org/wiki/images/6/68/T--KEYSTONE--Lcp2.png"></img src></div>
	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
−

---

Revision as of 15:31, 20 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.  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). 

Enzyme activity verification

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 

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.