Difference between revisions of "Part:BBa K3863008"

Revision as of 16:48, 20 October 2021

Phac-flag-PCT(optimized)-HA-phasin-his This is a composite part designed to produce bioplastic PHA. PHA synthase 1 from Pseudomonas resinovorans. The protein sequence in this part is based on the K2042001 sequence Evry 2016. propionate CoA transferase from Clostridum propionicum. The protein sequence in this part is based on the K1211001 sequence Yale 2013. Phasin attaches to the polyhydroxybutyrate (PHB) granule surface regulating the number and size of PHB granules within a cell. It probably also acts as a regulator affecting the biosynthetic activity of PHB synthase in vivo. Sequence and Features

Validation of construct expression of K3863008

Fig 1. Real-time qPCR fold change of reference gene

To future confirm that our constructs have the expression level, we have performed RT-qPCR The RT-qPCR results show that we have successfully assembled target genes K3863008 to the vector (pETDuet).

Validation of Bioplastic Production

In order to observe the conversion performance, infrared spectroscopic analysis was applied since PHA , PLA and PhaC have their particular functional group in chemical structure, and are performed in particular wavelengths. The absorption peak of PLA is at 1081, 1188, 1364, 1452 , 1751 cm-1[1], whereas the absorption peak 979, 1057, 1100, 1282, 1723, 2934, 2977cm-1[2]. We have compared and analyse the peak of the wavelength to confirm the bioplastic product we have produced. In Figure 2, no absorption peak of PHA and PLA was identified, which indicated that the vector (pETDuet Vector) cannot give the desired product. On the other hand, in Figure (3b) and (3c), the absorption peaks of PHA and PhaC were identified after incubation, which suggested that the BBa_K3863004 (in Figure (3b)) and BBa_K3863008 (in Figure (3c)), the bioplastic performs significantly in the transformation process. Furthermore, the absorption peaks of PHA in Figure (d) were found, which showed that BBa_K3863007 performs well and was the product that we expected to have. These results have proven that the bioplastic that we have produced can be formed significantly with BBa_K3863004, BBa_K3863008 and BBa_K3863007.

Figure 2a. IR spectrum of pETDuet vector

Figure 2b. IR spectrum of BBa_K3863008

Quantitative Measure of Bioplastic Production

In order to measure the amount of bioplastic produced, we used 20 microgram Nile Red (sigma aldrich N3013) per milliliter of agar to make a Nile agar plate and streak the cell culture (PHA, PhaC, Phasin) on the plate. After incubating for 2 days at 37 celsius in darkness. Stereomicroscope (Nikon SMZ18) is used to measure the intensity of Nile red.

Mean intensity of Nile red for quantitative measure of bioplastic production of (BBa_K3863007(PHA), BBa_K3863004(PhaC), BBa_K3863008(Phasin))

Reference

[1]https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3450024/pdf/12088_2009_Article_31.pdf
[2]https://www.researchgate.net/figure/Fourier-transform-infrared-FTIR-spectra-of-PLA-PEG-PLA-PEG-blend-and-PLA-PEG-xGnP_fig1_277675367