Difference between revisions of "Part:BBa K2593006"

Revision as of 01:21, 18 October 2018

PlepA-RBS-AmyX -6His-LHyal-T1

This composite part consists of a promoter, an RBS, a signal peptide from Bacillus origin (AmyX), a coding sequence (LHAases) with a 6-His tag fused to its N-terminus, and a terminator.
Biology
PlepA (BBa_K823002) is the promoter of the lepA gene of Bacillus subtilis. It is a constitutive promoter and does not contain a ribosome binding site.
PlepA is a strong constitutive promoter which was derived from the bicistronic operon. One of the expressed proteins in the operon is protein PlepA. This protein plays an important role during the translation as it can move the mRNA-tRNA complex one step back in the ribosome which is expected to improve the fidelity of translation.
RBS (BBa_K2593005):The strong ribosome binding site (RBS) is a shine-Dalgarno sequence from gsiB gene. It can lead to a pronounced stimulation of expression when placed upstream of a variety of genes, and significant increase in the translation of the genes is observed.
AmyX-6His-LHyal (BBa_K2593009):AmyX signal peptide: AmyX signal peptide is encoded by a-Amylase gene which is derived from B.amyloliquefaciens. AmyX signal peptide belongs to twin-arginine translocation (Tat) pathway of the Bacillus origin. The Tat pathway has a signature twin-arginine (RR) motif located at the border of the N-terminal domain and the hydrophobic region. The Tat systems of Gram-positive bacteria exhibit interesting differences to those of Gram-negative bacteria, the most striking of which is the absence of a TatB component in virtually all species. In Gram-negative bacteria, membrane-bound TatABC subunits are all essential for activity, whereas Gram-positive bacteria usually contain only TatAC subunits. In Bacillus subtilis, there are two TatAC-type systems, TatAdCd and TatAyCy, operate in parallel with different substrate specificities. in our project AmyX was fused in N-terminal of the LHyal gene for the extracellular export of the expressed protein.
6xHis: Currently His-tags have been extensively applied for recombinant protein expression. Based on previous research, 6His-tag could provide enough spacing for protein folding to prevent crystallization or misfolding of peptide chain, and it is a commonly used in protein purification by chromatography.
LHyal gene encodes LHAase (Mw=58kD), which is a hyaluronidases from the hyaluronate 3-glycanohydrolases sub-family of leech origin. Hyaluronidase (HAase) is a large family of glycoside hydrolases that predominantly degrades hyaluronic acid (HA), which is a polysaccharide composed of disaccharides unit of N-acetyl glucosamine and glucuronic acid polymerization.
T1 terminator(BBa_B0010): , it is the most used terminator in E. coli system.

Usage

In our project, this device worked to produce hyaluronic acid hydrolase (hyaluronidase) which is derived from leech genome.We measured the LHAase expression at transcriptional level(Fig 1). Given Figure 2 showing that LHAase production continued to rise up to 60 h, it is possible to assume that LHAase production may cease to increase after 60 h, due to a drop in mRNA transcripts after 48 h of bacterial growth. In the future we will explore this further.

Figure1: agarose gel electrophoresis of mRNA transcript at various time points. (1-7): 6h, 12h, 24h, 36h, 48h, 60h, NTC. Top strand showing RT-PCR products amplifying a section of LHAyal coding sequence, bottom strand showing RT-PCR products amplifying a housekeeping gene sequence (16s rRNA).

Figure 2: analysis of enzyme activity of recombinant LHAase and cell growth. A typical time course of recombinant LHAase expression in B. subitlis 168. LHAase enzyme activity (red, U/ml), and cell growth density (black, OD600 value) were measured at regular intervals.

In addition, in order to demonstrate the functionality of this part, we performed ELISA studies, enzymatic activity (DNS method and plate assay) to study the function of LHAase.

Figure 3: enzymatic activity of recombinant LHAase expressed from B.subtilis strains harboring pDG1730-AmyX-H6LHAyal or pMA0911-AmyX-H6LHAyal, analyzing by ELISA.

Figure 4: enzymatic activity of recombinant LHAase expressed from B.subtilis strains harboring pDG1730-AmyX-H6LHAyal or pMA0911-AmyX-H6LHAyal, analyzing by DNS reducing sugar assay.

Figure5 : Determination of LHAase activity using a HA plate assay, where cylindrical holes were injected with fermenting supernatant of LHAase-expression (pDG1730, pMA0911) or control (empty vector) B. subtilis 168. A positive control used commercial BTH, while a negative control used water. The white circles in pDG1730 and pMA0911 showing the color residues from fermenting culture.

T Sequence and Features

Reference

[http://www.ncbi.nlm.nih.gov/pubmed?term=Microbiology%2C%20142%3A%201641%E2%80%931649: Homuth et al., 1996], [http://www.ncbi.nlm.nih.gov/pubmed?term=Cell%2C%20127%20%284%29%3A%20721%E2%80%93733: Qin et al., 2006]
Sunghoon Park, Wolfgang Schumann. Optimization of the Secretion Pathway for Heterologous Proteins in Bacillus subtilis[J].Biotechnology and Bioprocess Engineering,2015:626-627
Peng Jin, Guocheng Du, Zhen Kang. High-yield novel leech hyaluronidase to expedite the preparation of specific hyaluronan oligomers[J].Scientific Reports, 2014 : 1-2
Jinpeng, Kangzhen, Biosynthesis of hyaluronan oligosaccharides and construction of DNA editing and assembly tools[D]Jiangnan University: Jinpeng,2016.25-27.