Team:Wisconsin-Madison/lemon

• Team

  • Team Members
  • Advisors
  • Sponsors

• Projects

  • Engineering Limonene Production in E.coli
  • Translational Coupling Cassette (TCC)

• Parts

  • Translational Coupling Cassette
  • Negative TCC Control
  • Positive TCC Control
  • Codon-optomized LIMS1

• Modeling

• Protocol

• Safety

• Human practice

Translational Coupling – an explanation

Why Produce Limonene?

In the beginning of the summer our team’s goal was to produce a molecule called limonene. All of our strains failed to produce limonene, and we were unsure of the cause. One hypothesis was that there was a problem in the translation of limonene synthase. To test this hypothesis, we used the translational coupling cassette to determine if limonene synthase was being translated. With this information, we hoped to determine why limonene wasn’t being produced.

The Mevalonate Pathway

The goal of this project is to engineer Escherichia coli to produce a compound called limonene. Limonene is a 10 carbon monoterpene and is found naturally in the oils of citrus fruits. It is used as a cleaning agent, solvent, food additive and is finding a place in new medical applications. Limonene also possesses the chemical properties of an ideal biofuel, sparking interest specifically in its application as a jet fuel due to its low freezing point. Currently, we are limited to extracting limonene directly from citrus fruits which prevents its collection in quantities large enough to be useful as a biofuel. E coli. could be used to produce limonene more effectively and efficiently.

Assays for the translational coupling cassette

The mevalonate pathway is a series of enzymes used to take Acetyl-CoA to 3-isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP through several chemical reactions. IPP and DMAPP are the building blocks for a family of molecules called Isoprenoids which is a large group of organic molecules with a wide array of functions. It was used in the Jay Keasling’s lab to create Amorphadiene, an antimalarial drug much cheaper and faster than was previously possible.

In this research, a strain of E. coli will be bioengineered to produce limonene by inserting the genes for the necessary biochemical pathways. Our strain contains the genes coding for the Mevalonate pathway ,a synthesized Geranyl Diphosphate Synthase (GPPS) gene, and a synthesized and codon optimized Limonene Synthase gene. The plasmid, pBba5c, contains the genes for the mevalonate pathway in an operon under a lac inducible promoter. During our research we used the Gibson cloning method to take out the ispA gene and put in the GPPS gene into the pBba5c plasmid, creating pBba5c-GPPS for better production of Limonene. This strain would theoretically be able to produce Limonene using common media.

The Production Assay

5 ml cultures of each strain (listed below) were grown overnight at 37 C in LB, normalized to an OD600=1 and diluted 1:100 into 40 ml cultures. They were then grown up to OD600=0.2 and the promoters in the pBba5C vector were induced with 1 mM IPTG. 10 mL of dodecane was placed over the top of the culture as an organic layer for the limonene to be captured. The cultures were grown for 18 hours, spun down, and 1mL of the dodecane overlay was diluted in ethyl acetate and sampled in a GC/MS.