Team:SZPT-CHINA/Parts/Overview

S Z P T - C H I N A

• Home
• Project
  • Description
  • Design
  • Modeling
  • Results
  • Demonstrate
  • Safety
  • Protocols
  • Notes
• Team
  • Teammates
  • Collaborations
  • Attributions
• Human Practices
  • Overview
  • Human Practices
  • Euducation & Engagement
  • Meet Up
• Parts
  • Overview
  • Characterization
  • Improved Parts
• Achievements

PARTS

INTRODUCTION

Our parts consist of three parts: (1) related to acid sensors; (2) related to the expression of antihypertensive peptides; (3) autolysis of lactic acid bacteria. All parts were generated during our project. There are 12 basic parts and 4 composite parts.

PARTS RELATED TO PH SENSOR

The rcfB promoter, a member of the CRP-FNR positive regulation family, was amplified from chromosomal DNA of L. Lactis subsp. lactis IL1403. It is an important part of the P170 promoter regulated by acid. When the external PH value declines to 5.5, rcfb promoter will be highly upregulated. [1]

Lac repressor (LacR) and operator (O1 and O2) sequences is part of the lactose operon derived from Lactobacillus delbrueckii. O1 and O2 may serve as operators for the binding of the lac repressor (LacR). [2]

Part Number	Name	Type	Part Description
BBa_K3142000	PrcfB	Basic	Acid-induced promoter
BBa_K3142001	O1/O2	Basic	Lactose operator
BBa_K3142015	LacR	Basic	Lac repressor
BBa_K3142002	PrcfB+LacR	Composite	Acid-induced promoter+ repressor LacR of lac operon

PARTS RELATED TO EXPRESSION OF ANTIHYPERTENSIVE PEPTIDE

Antihypertensive peptides (AHPs) are protein-derived short peptides released by hydrolysis processes, which act as inhibitors of angiotensin-converting enzyme and renin.

The 11S globulin from amaranth (A11Sg) is the most abundant storage protein in mature seeds and is well recognized for its nutritional value. We used this globulin to engineer a new protein by adding five highly active antihypertensive peptides at its C-terminal end to improve its functionality. [3]

Part Number	Name	Type	Part Description
BBa_K3142003	FR	Basic	Arginine Linker
BBa_K3142004	A11Sg	Basic	11S globulin from amaranth
BBa_K3142005	VY	Basic	Antihypertensive peptide
BBa_K3142006	IPP	Basic	Antihypertensive peptide
BBa_K3142007	VPP	Basic	Antihypertensive peptide
BBa_K3142008	KYLCY	Basic	Antihypertensive peptide
BBa_K3142009	FKGKYYP	Basic	Antihypertensive peptide
BBa_K3142010	AHPM	Composite	Antihypertensive peptide multimer
BBa_K3142011	A11Sg-AHPM	Composite	Complex of 11S globulin from amaranth and Antihypertensive peptide multimer

PARTS RELATED TO LACTIC ACID BACTERIA AUTOLYSIS

Enhanced truncated promoters (T-αcrp) which include a modified enhancer element from the CC(-41.5)α(-63) synthetic elements upstream of the T-crp promoters. Minimal “truncated” promoters (T-crp) containing ~ 50 bp upstream of the transcription start site with care taken to include the full CRP binding site. T-αcrp promoter can response to glucose starvation to Initiate expression of downstream genes. [4]

N-acetylmuramidase (acmA) from Lactobacillus acidophilus NCFM dissolves the cell wall of lactic acid bacteria, causing lactic acid bacteria to cleave. [5]

Part Number	Name	Type	Part Description
BBa_K3142012	PT-αCRP	Basic	Enhanced truncated promoters
BBa_K3142013	acmA	Basic	N-acetylmuramidase
BBa_K3142014	PT-αCRP + acmA	Composite	Enhanced truncated promoters+ N-acetylmuramidase

References

[1] Ismail AKYOL, Ugur COMLEKCIOGLU, Asuman KARAKAS,et,al. Regulation of the acid inducible rcfB promoter in Lactococcus lactis subsp. Lactis. Annals of Microbiology, 58 (2) 269-273 (2008)

[2] United States Patent . Patent No.US 6,929,931 B1. Date of Patent:Aug. 16, 2005

[3] Edgar Espinosa-Hernández , Jocksan Ismael Morales-Camacho , D. Alejandro Fernández-Velasco，et,al. The insertion of bioactive peptides at the C-terminal end of an 11S globulin changes the structural stability and improves the antihypertensive activity. Electronic Journal of Biotechnology 37 (2019) 18–24

[4] William Henry Bothfeld, Grace Kapov, and Keith Tyo. A glucose-sensing toggle switch for autonomous, high productivity genetic control. ACS Synth. Biol,Just Accepted Manuscript•DOI: 10.1021/acssynbio.6b00257•Publication Date (Web): 08 Mar 2017

[5] Pfeiler EA, Azcarate-Peril MA, Klaenhammer TR. Characterization of a novel bile-inducible operon encoding a two-component regulatory system in Lactobacillus acidophilus. J Bacteriol. 2007 Jul;189(13):4624-34. Epub 2007 Apr 20.