Part:BBa_K2739005

proR-PhaR-proP-PhaP

This is a composite part that was created to allow us to test the functionality of the phaR and phasin under the control of their native promoter.

Experiments and Results

Recombinant E.coli harbouring phaCAB operon + phaR autoregulation system with and without phasin were constructed to investigated the influence of phaR autoregulation system on PHB production and cell growth rate. Recombinant E.coli harbouring pSB1C3 + pSB3T5, PHA operon + Phasin, PHA operon + pSB3T5 were also investigated as the controls. 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. The protein fusion of phasin and HlyA was expressed for PHB secretion via Type I secretion system. The influence of HlyA depending secretion on PHB was also investigated.

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 858
  Illegal SapI.rc site found at 253