Team:Nanjing-China/Project

Overview

Exogenous RNAs are flowing and working in our body, and food carries them besides traditional nutrients. Evidence shows that natural plant miRNAs can be ingested into mammal bodies and target specific genes. Such discoveries show us a promising approach to perform cross-kingdom information transportation and gene regulation.

We propose a method of controlling animal gene expression and helping cure disease by creating vegetable that produces artificial siRNAs targeting critical genes for some disease. This time, we chose PGC-1 alpha gene, which is over-expressed in fatty liver and contributes to insulin resistance, as the target. This concept can provide us a better perception of our daily diet and a new way of curing disease.

On the other hand, we expand the boarder of iGEM by working on green plant. Firstly standardized binary vector is constructed. Also, we designed a brand device for transgenic plant to help solving potential safety problems.

Module :small RNA producing

Background

A recent study demonstrated that plant small RNAs acquired orally through food intake directly influence gene expression in animals after migration through the plasma and delivery to specific organs.[1][2] This fact indicates that we can use transgenic plant as a convenient small RNAs producer and a direct delivery system for therapeutic small RNAs .

Other study shows that high expression of PGC1-alpha in liver potentially contributes to systemic insulin resistance, glucose intolerance, and insulin deficiency.[3] Based on these two facts, we are able to develop a small RNA producing vegetable to turn down PGC1-alpha expression, and thus help rescue impaired insulin sensitivity in patience with Type II diabetes.

Motivation

This method would be a great opportunity of synthetic biology in both food and medicine. Instead of screening for drugs from thousands of small molecular candidate which might bring unknown side effects. Developing therapeutic si RNA through food intake is a lot faster and safer. On the other hand, our part would be transform into other artificial small RNA generator easily.

Part designs

pWe designed a siRNA for PGC1-alpha, and put it in a plant endogenous miRNA gene[4] from Arabidopsis thaliana, and made it BBa_K788002 in the registry. To ensure its high and constant expression in plant, we chose CamV 35S promoter and NOS terminator. We made BBa_K788000 and BBa_K788001 from a commonly used plant expression vector pBI 121.

Method

When finish assembly of CamV 35S promoter and ami-RNA generator and NOS terminator, we use pPZP 212(BBa_K88004), a binary vector for plant transfection, with Kanamycin resistance. Then we used bacterium to finish the transfection (details plz see Note section). After selection, we can get the plant we want.

Measurement

The transfection can be measured by Kanamycin selection tissue culturing. And we used RT-PCR by special designed primers to confirm the expression of the small RNA we want.[5]

Result

One of the results of the selection

And the RT PCR has confirmed the expression of artificial micro RNA for PGC1-alpha

Module Binary Vector

Motivation

Although pPZP 212 and other Agrobacterium binary vectors are very useful, the philosophy of BioBricks inspire us to consider more about this. We want to design parts that can be easily use for iGEMer to contruct their own binary part. Just image if some future team need to create bacteria symbiosis with plant express its own Parts and send genes to the host plant at the same time.

Part designs

T-DNA segment( including LB and RB) is an Agrobacterium transfection element. Sequence between two T-DNA segment will be transferred to host plant’s genome. We design LB part (BBa_K788010), RB(BBa_K788011) part. Besides there is another DNA segment containing Mun I and Eco T22 I digestion sites which can do ligation with the EcoR I and Pst I site in all standardized plasmid backbone (BBa_K788005). More Detail of their designs in on the parts registry.

barcode2

Motivation

Biosafety issues raised by plant Synthetic Biology can be partly solved by better documentation within a BioBricks containing organism and more responsibility. Besides the original barcode after the stop codon, we need a longer and clearer signature which contains the information of constructor, date, and other information about the artificial system inside.

construct

We set up simple rule for Barcode 2 parts for each system A/C for 1 and G/T 0, 6 binary numbers is one set. And their interpretation follow special rule. We put “NJ 12”in the BBa_K788007 part. More detail is on the registry.

parts

More detail and discussion can be seen in Human Practice Bio safety section and partregistry BBa_K788007.

Reference

• [1] Ingested plant miRNAs regulate gene expression in animals, Hervé Vaucheret, Yves Chupeau, Cell Research (2012).
• [2] Exogenous plant MIR168a specifically targets mammalian LDLRAP1: evidence of cross-kingdom regulation by microRNA, Lin Zhang et al, Cell Research (2011) .
• [3] PGC-1 coactivators: inducible regulators of energy metabolism in health and disease, Brian N. Finck,  J. Clin. Invest. 116:615–622 (2006). doi:10.1172/JCI27794
• [4] Directed Gene Silencing with Artificial MicroRNAs, Rebecca Schwab et al, B.C. Meyers and P.J. Green (eds.), Plant MicroRNAs, Methods in Molecular Biology, vol. 592.
• [5] Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs Erika Varkonyi-Gasic, Plant Methods 2007