Team:KEYSTONE/Proof Of Concept

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• Overview
• A efficient Lcp——Lcp1 _VH2
• More Lcp1 _VH2
• Lcp1 _VH2 Secretion

Overview

The aim of the project is to utilize genetic engineering methodologies to acquire a biological product that can degrade rubber (cis1,4-polyisoprene) effectively. The critical agent Latex Clearing Protein (Lcp), an enzyme that can break down the chain structure of polyisoprene and reduce it to its monomeric form: isoprene. Aiming to improve the use of in a biological system,we have undertaken additional designs to facilitate the process in which Lcp is produced and put into practice.

A efficient Lcp——Lcp1 _VH2

First of all, we utilized Lcp1 _VH2, a new part of iGEM to tackle rubber degradation. Lcp1 _VH2 can effectively achieve heterologous expression in E.coli BL21, and has the enzyme activity of rubber degradation. In addition, we tested an old brick Lcp_K30 from these two aspects. AS shown in Fig1A, Lcp1 _VH2 has a better expression than Lcp_K30. Lcp belongs to the oxygenase family, which utilizes oxygen to break down carbon double bonds. Therefore, we designed an oxygen concentration experiment to determine the enzyme activity of the two Lcps. The oxygen in the sample tube (Supernatant containing Lcp1 _VH2) is consumed more quickly until 0, which suggests Lcp1 _VH2 has a higher enzyme activity.

Fig. 1 A. SDS-PAGE of crude extracts(C) and soluble fractions(S). BL21 as control (1), Lcp1 _VH2 (2), and Lcp_K30 (3). B. The comparison of the activity of Lcp1 _VH2 and Lcp _K30 using an oxygen concentration test.

More Lcp1 _VH2

Secondly, we improved the yield of Lcp1 _VH2 by adding a fusion partner. From literature, we found the addition of a solubility-improving fusion protein named NusA to the N terminal of Lcp1 _VH2, an enzyme with low solubility, may be a viable way to improve its yield. Surprisingly, experimental results showed Lcp1 _VH2 was adequately soluble in Fig. 2A, the concentrations of Lcp1 _VH2 in crude extract and the supernatant are similar. In general, The addition of NusA boosted the yield of Lcp1 _VH2. We believe this situation is caused by NusA abating the cellular toxicity of the Lcp1 _VH2. We examined the activity of the enzyme by testing the dissolved oxygen in crude extract and supernatant in which the substrate, granular cis1,4-polyisoprene was added. The results, in Figure 2B, showed that the improvement in yield did not come with an increase in activity. In hopes of overcoming this obstacle, we interviewed Professor Liu from BUCT [url], from which we learned that full enzyme activity can be restored by cleaving off NusA after it has been secreted. In conclusion, the addition of NusA improved the yield of Lcp1 _VH2 and broadened the potential of the project’s implementation.

Fig 2. A. SDS-PAGE of crude extracts(C) and soluble fractions(S) of E.coli BL21(1) as control, Lcp1 _VH2 (2) and fusion proteins NusA-Lcp1 _VH2 (3), the first box shows the expression of Lcp1 _VH2 and the second box shows that of NusA-Lcp1 _VH2; B. The oxygen dissolving test conducted on Lcp1 _VH2 and NusA-Lcp1 _VH2

Lcp1 _VH2 Secretion

The last modification is the addition of signal peptides to simplify the fermentation process. We wish to achieve extracellular secretion of Lcp by combining it with signal peptides on its terminus. After trials of selection, we have identified an effective signal peptide, HlyA, using laccase as the reporter as it can react with ABTS dissolved in the media. From the experiment, as shown in Figure 2A, the secretion of Lcp is clearly indicated by laccase, but we also found that laccase by itself can be a secretion signal. Therefore, we designed the plasmid pET23a-laccase-Lcp1 _VH2-HlyA as our final product, we expressed it in E.coli BL21. AS shown in Fig 3C, the culture fluid is the metabolites of E.coli BL21(including the secreted Lcp1 _VH2), the supernatant is the soluble Lcp1 _VH2 after lysis and centrifugation. The results show Lcp1 _VH2 in both supernatant and culture medium displayed enzyme activity, Although the concentration of Lcp1 _VH2 appears to be higher in the supernatant than in the culture medium. We can prove that Lcp1 _VH2 autonomously secreted by E.coli BL21 has enzymatic activity.

Fig. 3 A. Coloration experiment using medium containing ABTS; B. SDS-PAGE of crude extracts(C) and soluble fractions(S) of E.coli BL21(1) as control, Lcp1 _VH2 (2) and fusion proteins Laccase-Lcp1 _VH2-HlyA (3); C. Oxygen consumption reaction using supernatant and culture fluid of E.coli BL21 pET23a::laccase-Lcp1 _VH2-HlyA.

Overall, the results we have obtained aligned with most of our assumptions. The improvements in yield and the capacity of extracellular secretion will make Lcp1 _VH2 more suitable for industrial-scale implementation.