Latest revision as of 01:12, 18 October 2018

proR-PhaR-proP-PhaP-HlyA

This is a composite part that was created to allow us to test the functionality of the HlyA signal peptide under the coexpression of the phaR and phasin, which are under the control of their native promoters. PhaR is the core member of the phasin autoregulation system, which contribute to the PHA operon and PHA production. It is known to repress the expression of the phasin through binding to the phasin promoter and auto-regulate its own expression. Otherwise, phaR can also bind to the PHB granules. With the coexpression of the PHA operon in recombinant E.coli, this part has verified its novel function in improving the PHA production and also PHA secretion.

Usage and Biology

The protein fusion of phasin and HlyA was expressed for PHB secretion via Type I secretion system.

According to previous research (Pötter et al, 2002), phaR binds at two sites upstream of phasin. One site is the transcriptional initiation site plus the -10 region. The other site is a region just upstream of the -35 region of the σ70 promoter of phaP. Gene of phaR was also recognised to bind to 86 bp upstream of the start codon in the open reading frame (ORF) of phaR. In addition, PhaR was detected binding on the surface of PHA granules (Pötter et al, 2002; Yamada et al, 2013). Based on the binding behaviours and following influence, it was confirmed that phaR regulated not only phaP but also itself precisely (Pötter et al, 2002).

During the later stages of PHA accumulation, PhaR leaves the PHA granules as the surface of PHA granules is mostly covered with phasin. The replaced PhaR returns to bind to the phaP promoter and represses transcription of phasin again. PhaR has been reported to autoregulate the expression of itself by binding on its promoter(Yamada et al, 2013).This autoregulation system guarantees the efficient expression of phasin as well as curtailing excessive expression of the PHA biosynthetic pathway, decreasing the metabolic burden imposed on cells.

Experiments and Results

Recombinant E.coli harbouring phaCAB operon + phaR autoregulation system + Phasin fused with HlyA signal peptide were constructed to investigated the influence of phaR autoregulation system on PHB production, secretion and cell growth rate. To allow the comparison, several related E.coli EL(21) DE3 strains were also developed, these include PHA operon + Phasin, phaCAB operon + phaR autoregulation system + Phasin, pSB1C3 + pSB3T5, PHA operon + pSB3T5 and investigated. Herein, the phasin is controlled by its native promoter, isolated from R.eutropha. Furthermore, HlyA depending secretion was reported to function in recombinant E.coli for PHB secretion.

Figure 1. The sequence alignment of BBa_K390501, phaR-Phasin-Hly-VR(The sequencing result of our modified part]) and original-phasinHlyA (The sequencing result of BBa_K390501) via Benchling. The selected part of the sequence indicated the stop codon.

The effect of PhaR on cell growth

In order to investigate the effect of produced protein PhaR on cell growth, cells that harboured pSB1C3 + pSB3T5, PHA operon + Phasin or pSB3T5 or phaR (R) or phaR-phasin (R-P) or phaR-Phasin-HlyA (R-P-H) were cultivated in M9 medium with 3 % glucose to plot the growth curve (figure 1). E. coli strain BL21 (DE3) harbouring PHA operon + phaR (R) or phaR-phasin (R-P) or phaR-Phasin-HlyA (R-P-H) reached the top three optical density after 30 hours of cultivation and their maximum optical densities were estimated to be approximately OD600 7.5, 5 and 4.2.

Figure 1. The growth curve of recombinant E.coli strains.

Comparing the PHA production of E. coli expressing new construct

To confirm that the cell harbouring new constructs indeed produce PHA, those recombinant E. coli BL21 (DE3) strains was spread on the Nile red agar plates with negative control (pSB1C3) respectively, and plates were exposed to blue light. In each picture of a, b, c, d, e, there were 4 Nile red plates. The left plates on the first rows were strains harbouring phaCAB operon and constructs. The right plate on the first row were strains containing the construct only. The second row were strains harbouring phaCAB operon only as well as pSB1C3and pSB3T5 backbones. phaCAB operon strain showed strong signal of fluorescence because of PHB produced and the backbone strain did not due to no PHB produced.

Figure 2. . Nile red plates culture to confirm PHB production (48 hours).

PHA Extraction and Melting temperature measurement

After the Nile red fluorescence detection, different strains of recombinant E.coli were proven to becapable to produce PHB. These six E.coli BL21 (DE3) strains were then extracted after incubated in M9 medium and 3% glucose for 72 hours. As the yield was too low to weigh the extracted PHB power accurately even on an analytical balance, the extracted PHB product from triplicated culture were mixed together to quantify.

Table 1. The yield of PHB production

Figure 3. Data for dry weight of intracellular and secreted PHB.

Due to the difficulty in access GC for PHA product analysis, the melting temperature of the products were measured. As a characteristic property of the substance, melting temperature is usually reported as a temperature range between the points where melting starts and is completed. To compare the purity, the pure PHB was borrowed from other lab to test melting point, getting the result of 160-170°C. Checking from references, melting temperatures of pure PHB are slightly different in distinct sources, ranging from 165-180°C (Porter and Yu, 2011; Owen et al, 1992). Compared with pure PHB, the bacteria produced product started to melt at a little bit lower temperature.

Figure 4. Melting temperature of PHB produced by recombinant E.coli strains.

Sequence and Features