Difference between revisions of "Team:British Columbia/Project/Bio-Pathways"

Abstract

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Key Achievements

Introduction

Escherichia coli is commonly used as a chassis for the biosynthesis of valuable chemicals. Advantages to its use include fast growth kinetics, ease of acquiring high density cultures, ability to grow on many types of media that can be made from inexpensive reagents, and tractability for genetic manipulation (Rosano and Ceccarelli 2014). As such, many past iGEM teams have chosen to engineer biosynthetic pathways into E. coli for production of a wide range of commercially-desired products such as biofuels, violacein, astaxanthin, and its precursor β-carotene.

This year, we intend to build a platform for producing valuable chemicals more sustainably by bridging lignocellulose processing directly to product manufacture. For our project, we decided to engineer E. coli to produce β-carotene in tandem to its growth with C.crescentus. In particular, we designed our system so that E. coli metabolizes the simple sugars released from cellulosic degradation accomplished by cellulase-expressing C. crescentus, to in turn produce β-carotene as a secondary metabolite.

β-carotene is a carotenoid found in many colored fruits and vegetables. It is the biosynthetic precursor to vitamin A which has roles in gene expression, vision, maintenance of body linings and skin, immune defenses, growth of the body, and normal development of cells (Sizer and Whitney 2016). Aside from being a precursor, β-carotene also has functions as a potent quencher of singlet toxic oxygen species, and can act as an antioxidant that scavenges free radicals in human low density lipoprotein (LDL), high density lipoprotein (HDL), and cell membranes (Bendich 2004).

We chose to produce β-carotene as a proof-of-concept approach to validating the functionality of our system. However, our platform can be applied to the production of higher value chemicals as well. We anticipate that directly linking chemical production to treatment of lignocellulosic biomass, in the form of a microbial consortium, would result in both effectively reducing the costs associated with chemical production and valorizing lignocellulosic waste.

Design

WE MUST ADD THIS SECTION :)

Methods

Growth of DH5α E. coli cells expressing β-carotene in minimal medium

Chemically competent DH5α E. coli cells were transformed with the 2009 Cambridge iGEM team’s construct, BBa_K274210, which encodes an operon for β-carotene production using pyruvate and glyceraldehyde-3-phosphate as substrates. Transformed cells were plated onto LB agar + CM plates. Isolated colonies with yellow-orange color were then inoculated - in triplicates, in LB + CM, as overnight cultures to be used in subsequent experiments for growth curve generation. Likewise, untransformed DH5α E. coli cells that did not produce β-carotene were also inoculated - in triplicates, in LB+CM, as overnight cultures that would serve as a negative control.

To generate a curve for growth in minimal medium containing varying concentrations of glucose, overnight cultures of DH5α E. coli cells that either express or do not express β-carotene were inoculated to a startin goptical density (OD600) of 0.01 - in triplicates, into 100ml of M9 minimal medium containing 100 μg/ml ampicillin, and either 0.05, 0.10 or 0.20% glucose. OD600 was subsequently measured at different times over the course of approximately 50 hours using a spectrophotometer.

To generate a curve for growth in minimal medium containing cellobiose or cellulose, overnight cultures of DH5α E. coli cells that either express or do not express β-carotene were inoculated to a starting optical density (OD600) of 0.01 - in triplicates, into 100ml of M9 minimal medium containing 100 μg/ml ampicillin, and either 0.20% cellobiose or 0.20% cellulose. OD600 was subsequently measured at different times over the course of approximately 50 hours using a spectrophotometer.

Characterizing β-carotene production in DH5α E. coli cells β-carotene biosynthesis gene

Cloning CrtEBIY under a constitutive promoter into pSB1C3 plasmid

All Escherichia coli cultures were grown in Luria Broth (LB) media supplemented with 12.5 μg/ml chloramphenicol (CM) unless stated otherwise. All plasmid DNA extractions were performed with QIAprep Spin Miniprep Kit (Qiagen). DNA purification from gels or PCR mixtures were done with NucleoSpin® Gel and PCR Clean-up kit (Macherey-Nagel).

DH5α E. coli cells encoding β-carotene under the BBa_R0011 constitutive promoter in pSB1A2, was PCR-amplified from BBa_K274210 using the VF2 (5’ - TGCCACCTGACGTCTAAGAA -3’) and VR (5’ - ATTACCGCCTTTGAGTGAGC - 3’) primers. The CrtEBIY amplicons were then gel purified, digested with EcoRI and SpeI, and ligated to psB1C3 plasmid that was also digested with EcoRI and XbaI, as well as gel purified. The ligation mixture was then transformed into chemically competent K12 (MG1655) and DH5α E. coli, and transformed cells were plated onto LB agar + CM. Several transformants were grown overnight in LB+CM, subjected to miniprep, and extracted plasmids were sent for Sanger sequencing using the VF2 primer for confirmation of the correct insert.

Results

Conclusion

References