Difference between revisions of "Team:UESTC-China/design"

Considerations

Considering the problem of durable TCP in the soil and combining the article degrading TCP into glycerol, we are intended to create a phytoremediation system degrading TCP constantly[1]. As the wild type of DhaA and HheC have low activity, in order to improve the degradation efficiency, we found the mutant Dha31[2] and HheC-W249P[3] by literature. Therefore, we decided to use Dha31, HheC-W249P and EchA to degrade TCP to glycerol. As well as this, we also need to consider that what kind of strategies could be suitable for the multigene co-expression? How can we achieve the three target proteins to accumulate stably in the tobacco? What methods can be used for improvement of protein expression?

Basic Design

In the expression vector design, we use the pGSD series of plant expression vectors and select 35S constitutive promoters from cauliflower mosaic virus (CaMV) to express our three enzymes; Golden Gate are used for DNA assembly. Besides these, to achieve the effective transmission of multiple foreign genes, several kinds of 2A peptide[4], from foot-and-mouth disease virus (FMDV) and some other picornaviruses, are fused to HheC-W249P, EchA and resistant gene, which acts as an autonomous element, making it an important tool for co-ordinated synthesis of multiple proteins from one open reading frame and making a ribosome jumping.

Single Gene Vector

For the purpose of achieving TCP degradation step by step，we start our project with three single gene vectors, piGEM2016-001,piGEM2016-002 and piGEM2016-003 including DhaA31,HheC-W249P and EchA respectively ,which are used for preparatory protein expression and degradation testing.

Multi-gene Vector

To make the ultimate goals of convert TCP pollutions to glycerol, we design a complicate vector piGEM2016-004 including DhaA31, HheC-W249P and EchA.

A Little Guess

Epoxide hydrolases, converting epoxides to diols, are widely distributed in the various species of mammals, insects, fungi, bacteria and plants. A role for Epoxide hydrolases in plants would be to break down epoxides accumulating during stress into less reactive compounds[5]. Furthermore, it’s reported that NtEH1, a kind of cytosolic Epoxide hydrolase, was found in the Nicotiana tabacum, which was expressed only in TMV-resistant tobacco after infection[6]. Hence, it’s suspected that there may be stable expression of epoxide hydrolases or inducible by epoxides stress in the Nicotiana tabacum. To verify this suppose, we design a two genes vector system piGEM2016-005 excluding EchA.

Optimized Design

Based on basic design, we hope to further optimize the vector expression system and make use of plant roots for better TCP degradation. Firstly, we use a root-specific promoter termed as PYK10[7] to enhance root-specific expression of protein. Secondly, a root-enhancing gene[8] is used to enhance the development of plant root systems. Thirdly, there is a signal peptide named by AOS[9] that can optimize the gene expression.

PYK10：root-specific promoter

Considering that TCP is mainly present in the soil and groundwater, root-specific expression may make it easier for TCP to be contacted with the enzyme. In addition, the expression of the three enzymes is limited to the roots, which, to a certain extent, play a role in enrichment. Therefore, we hope to use a root-specific promoter to express three target genes. After seeking hard, the root-specific promoter pYK10 from Arabidopsis thaliana is selected and piGEM2017-024 is designed.

CKX3：cytokinin-degrading cytokinin oxidase 3

The plant cytokinin is supposed as a kind of essential regulator of development of plant root systems. It’s reported that cytokinin-degrading cytokinin oxidase (CKX) is able to enhance root systems and get larger root through reducing the plant innate cytokinin status[8]. In CKX gene family, CKX3 effect is most outstanding. In order to improve target enzyme’s expression in the root, we plan to overexpress the CKX3 gene in tobacco. However, due to the expression of CKX in the whole plant plant will affect the growth and development of plants[8], so we select the PYK10 promoter to express CKX3 in the root. Ultimately, we built piGEM2017-021 using PYk10 and CKX3.Also, we design another vector named by piGEM2017-025 for control.

AOS：N-terminal ER-targeting signal peptide

AOS, an endoplasmic reticulum-targeting signal peptide from cucumber ascorbate oxidase, has great importance in protein expression. It’s reported that target protein can be modified with high mannose-type N-linked oligosaccharide(s) when the signal peptide is fused to protein[9], which is essential for protein stable accumulation in the plants cell. Furthermore, by fusing it to protein, the protein will be targeted at the apoplastic region, mainly cell walls[9], which is good for TCP degradation by means of plant roots. In order to avoid the phenomenon of transgenic silencing and improve the degradation effect of tobacco roots, we fuse AOs to three target genes. Accordingly, we constructed piGEM2017-014, piGEM2017-015, piGEM2017-016.

Combine all three strategies, we design two complicate vectors system termed as piGEM2017-022 and piGEM2017-022.

GUS Staining Verification

For easily visualizing the expression of target genes in Nicotiana tabacum, we constructed recombinant plasmids, in which a GUS gene was cloned after the three target genes.

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