Difference between revisions of "Part:BBa K3993013"

Revision as of 02:35, 20 October 2021

PPGK1-AsADC-TADH1 - PTEF1-SPE1-TTEF1 - PTDH3-speB-TCYC1

Profile

Name: PPGK1-AsADC-TADH1 - PTEF1-SPE1-TTEF1 - PTDH3-speB-TCYC1

Base Pairs: 6866 bp

Origin: Saccharomyces cerevisiae, E. coli, synthesis, genome

Properties: Arginine metabolism and polyamine biosynthesis chemical reactions

Usage and Biology

Kl tropane alkaloids (TAs) refers to a kind of alkaloids containing the tropane alkyl skeleton formed by the combination of pyrrole ring and piperidine ring in structure. It is a natural product of plant and has a long history and important medicinal value. tropane alkaloids have great market demand and often appear in global shortages. A method that can produce Tas in scale is expected. Using synthetic biology to create a microbial cell factory to produce TAs is a highly potential strategy.

The Tropane alkaloid (TAs) is obtained by a series of chemical reactions through the formation of Putrescine (1, 4-butylenediamine, Putrescine) from Arginine. Putrescine is an essential polyamine for ribosomal biogenesis and mRNA translation, but is regulated by polyamines and remains at low concentrations during normal cell growth. In this study, by overexpressing the natural genes involved in arginine metabolism and polyamine biosynthesis, the regulatory mechanism of polyamine biosynthesis is adjusted, so as to engineer the production of excessive putrescine strains.

Figure1. Principle diagram of TAs..

Construct design

The Tropine part of Tropane alkaloids (TAs) is obtained from arginine to putrescine (1,4-butanediamine, putrescine), and then through a series of chemical reactions. In this project, natural genes involved in arginine metabolism and polyamine biosynthesis was designed to overexpress in yeast. The engineer strains that produced excess putrescine. (Figure 2).

Figure 2. DNA sequence map of plasmid pYES2-AsADC-SPE1-SpeB..

The profiles of every basic part are as follows:

BBa_K3993000

Name: SPE1

Base Pairs: 1401bp

Origin: Saccharomyces cerevisiae, genome

Properties: Catalyzes the first and rate-limiting step of polyamine biosynthesis that converts ornithine into putrescine

Usage and Biology

his protein is involved in step 1 of the subpathway that synthesizes putrescine from L-ornithine. Catalyzes the first and rate-limiting step of polyamine biosynthesis that converts ornithine into putrescine, which is the precursor for the polyamines, spermidine and spermine. Polyamines are essential for cell proliferation and are implicated in cellular processes, ranging from DNA replication to apoptosis. Homodimer and only the dimer is catalytically active, as the active sites are constructed of residues from both monomers

BBa_K3993001

Name: SPEB

Base Pairs: 921bp

Origin: E. coli, genome

Properties: Catalyzes the formation of putrescine from agmatine.

Usage and Biology

This protein is involved in step 1 of the subpathway that synthesizes putrescine from agmatine. This subpathway is part of the pathway putrescine biosynthesis via agmatine pathway, which is itself part of Amine and polyamine biosynthesis. The expression of AUH activity is antagonistically regulated by cyclic AMP and agmatine. In the presence of the cAMP receptor protein, cAMP represses the expression of AUH, while agmatine induces it.

BBa_K3993002

Name: AsADC

Base Pairs: 1821bp

Origin: Saccharomyces cerevisiae, synthesis

Properties: codon optimized SPE1

Usage and Biology

This protein is involved in step 1 of the subpathway that synthesizes agmatine from L-arginine. This subpathway is part of the pathway agmatine biosynthesis, which is itself part of Amine and polyamine biosynthesis.

BBa_K3993003

Name: PTDH3

Base Pairs: 673bp

Origin: Addgene

Properties: Yeast centromeric vector with the TDH3 (glyceraldehyde 3-phosphate dehydrogenase) promoter.

Usage and Biology

Yeast CEN/ARS vector (Leu2) that contains multiple cloning site ( MCS ) and TDH3 promoter.

BBa_K3993004

Name: TCYC1

Base Pairs: 242bp

Origin: Saccharomyces cerevisiae, genome

Properties: CYC1 terminator

Usage and Biology

This is a common transcriptional terminator. Placed after a gene, it completing the transcription process and impacting mRNA half-life. This terminator can be used for in vivo systems, and can be used for modulating gene expression in yeast.

BBa_K3993005

Name: PPGK1

Base Pairs: 502bp

Origin: Addgene

Properties: A Yeast Expression plasmids backbone

Usage and Biology

Selection for in-frame fusion expression constructs.

BBa_K3993006

Name: TADH1

Base Pairs: 328bp

Origin: Saccharomyces cerevisiae, genome

Properties: ADH1 terminator

Usage and Biology

This is a common transcriptional terminator. Placed after a gene, it completing the transcription process and impacting mRNA half-life. This terminator can be used for in vivo systems, and can be used for modulating gene expression in yeast.

BBa_K3993007

Name: PTEF1

Base Pairs: 502bp

Origin: Saccharomyces cerevisiae, genome

Properties: A constitutive promoter

Usage and Biology

TEF1 gene have a strong promoter activity, and it has been shown to be constitutively expressed even in the presence of glucose. The TEF1 promoter could be used for production of homologous or heterologous proteins under conditions where the expression of a large number of genes involved in the use of less favoured carbon sources are repressed.

BBa_K3993008

Name: TTEF1

Base Pairs: 476bp

Origin: Saccharomyces cerevisiae, genome

Properties: TEF1 terminator

Usage and Biology

TEF1 terminator is a common terminator in yeast.

Experimental approach

1. Fragments PCR products Electrophoresis

Figure 3. Gel electrophoresis of amplified fragments..

Lane 1 is promoter PPGK1, Lane 2 is terminator TADH1, and Lane 3 is promoter PTEF1, Lane 4 is target gene SPE1, Lane 5 is terminator TTEF1. Lane 6 is promoter PTDH3, Lane 7 is the target gene AsADC, Lane 8 is the target gene speB.

2. overlap PCR to assembly the promoter-gene-terminator fragments

Figure 4. Gel electrophoresis of amplified fragments..

Lane 1 : promoter PPGK1 + the target gene AsADC , named seg1; Lane 2 : terminator TADH1 + promoter PTEF1, named seg2; Lane 3 : target gene SPE1 + terminator TTEF1. named seg3; Lane 4 : promoter PTDH3 + target gene speB , named seg4;

To do the 3rd round of overlap-PCR using the fragments recycled in figure4 as templates.

Figure 5. Gel electrophoresis of amplified fragments..

Lane 1 : seg1 + seg2 , named frag1; Lane 2 : seg3 + seg4 , named frag2;

To increase the yield of frag2, we performed the 4th round overlap-PCR.

Figure 6. Gel electrophoresis of amplified fragments..

Lane 1 to 3 is frag2; Lane 4 to 6 is terminator TCYC1; Lane 7 to 9 is the backbone plasmid pYES2.

To make up the long fragments, we performed the 5th round overlap-PCR.

Figure 7. Gel electrophoresis of amplified fragments..

Lane 1, 3, 5 : frag2 + terminator TCYC1; Lane 2, 4, 6 : seg4 + terminator TCYC1,named half1. This figure shows that the overlap PCR of seg4 and terminator TCYC1 was successful, while frag2 + terminator TCYC1 was not successful. So the 6th round of overlap-PCR was performed to overlap frag2+half1.

Figure 8. Gel electrophoresis of amplified fragments..

Lane 1 : frag2 + half1; In order to obtain our target plasmid, there are multi-fragment assembly plan A and B. In plan A, PCR amplification products of frag1, frag2, half1 and the backbone plasmid pYES2 were recovered from gel and the corresponding recombinant plasmids were transformed into competent cells for resistance screening of kana. In plan B, the sequence fragments were frag1, frag2, terminator TCYC1 and the backbone plasmid pYES2.

Figure 9.Verification of the plasmids via colony PCR..

Five single colonies in plan A and one single colony in plan B were picked up for further cultivate and plasmid extraction. Identification by electrophoresis showed that plasmid 1,4,5 of plan A and 6 of plan B were the candidate with correct size, in which plasmid 5 of plan A was confirmed by sequencing (Figure 9).

Sequence and Features