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Team:Berkeley/Project/GT
From 2013.igem.org
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| Indigo is so insoluble in water that it is not capable of dyeing clothes by itself. Industrial dyeing gets around this problem by reducing indigo to leuco-indigo, a white soluble version of the dye. In our project, we have found a way to use indican (a natural occurring compound in indigo plants) to dye clothing. Indican is a soluble precursor to indigo, and serves as a biosynthetic alternative to leuco-indigo. |
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Indigo producing plants have a naturally encoded pathway to produce indican. They utilize a glucosyl transferase (GT) to add a glucose molecule to the hydroxyl group of indoxyl. We intend to produce indican by co-expressing FMO and a GT in E. coli. Unfortunately, no sequence data is available for the glucosyl transferases that have activity on indoxyl. As part of our summer project, we embarked in a quest to find an indican producing GT. |
Glucosyl transferases(GTs) are ubiquitous and have been shown to act on a vast variety of substrates. As part of our search for an indican producing GT we have tested enzymes shown in the literature to have activity on substrates that resemble indoxyl.
This summer we have cloned a variety of GTs that have activity on compounds like benzoate, and jasmonate. A table with accession numbers and native substrates of our GTs is shown below.
As a first experiment, we attempted to express all of our GTs in E.coli to check for solubility. We decided to test solubility by generating a C-terminus fusion of GTs with a yellow fluorescent protein (Venus). A sample parts plasmid is shown below. Imaging under a fluorescent microscope showed that most of our GTs formed inclusion bodies (Represented by punctate fluorescence as opposed to diffuse fluorescence).
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This summer we have paid close attention to a bacterial glucosyl transferase (OleD). OleD is known for its broad substrate specificity, and we have cloned it to co-express with the indoxyl producing enzyme FMO. In addition, we have purified OleD to conduct in-vitro testing for the production of indican as well as other glucosides. OleD has been shown in the literature to further broaden its substrate specificity after introducing a set of active site mutations known as ASP mutations (A242V-S132F-P67T). Thorson et.al, 2010. We have recently cloned OleD-ASP and will be testing its activity on indoxyl as well as other substrates. |
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A very exciting approach at finding an indican producing GT involves going after those already encoded in Indigo Plants. This summer we have acquired 4 different indigo plants (Indigofera suffruticosa, Indigofera tinctoria, Polygonum tinctorium, and Isatis tinctoria), and used them to extract RNA from the leafs. RNA was reverse transcribed to generate cDNA libraries of all four plants. These cDNA libraries are being screened for indican producing GTs. In order to screen cDNA, we have generated a multiple sequence alignment of Glucosyl Transferases (See image below). |
Cropped version of a Multiple Sequence Alignment (MSA) of 122 B- UDP Glucosyl transferases found in the taxonomic group Core Eudicotyledons.
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This alignment revealed a well conserved region which we have used to make degenerate primers for PCR. Our screening efforts have started to give good results! We have extracted 3 glucosyl transferases that have never been studied before from indigo producing plants. These new GTs will be submitted to NCBI and characterized. |
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Gantt , Richard W. "Probing the Aglycon Promiscuity of an Engineered Glycosyltransferase." 47.46 (2008): 8889–8892. Print. |
