<p>Synthetic biology envisions a bioengineering domain for designing new genetic parts and systems, or redesigning of existing ones. It has employed the design-build-test-learn iterative cycle for ushering major advancements in the fields of bioprocess engineering and bio-therapeutics. Until now, the most versatile workhorse of synthetic biology has been Escherichia coli. However, there is a need for exploring new chassis which can be naturally adapted to unique traits or metabolic pathways. </p>
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<p>Lactococcus lactis (L. lactis) is a ‘Generally Regarded As Safe’ (GRAS) organism. It is naturally found in milk products and is also known to colonize the human gut. Unlike E. coli, it does not have a lipopolysaccharide (endotoxin) layer, which requires extra measures for purification. The range of products it can be engineered to produce include lactic acid (for bioplastics), biofuels (ethanol, butanol), biopolymers (PHAs), polyols (mannitol, sorbitol), and food flavors (diacetyl). Due to its ability to use a wide range of substrates and tolerance for a wide range of conditions (pH, temperature, solvent concentration), L. lactis serves as an alternate bacterial model for metabolic and bioprocess engineering. However, the absence of genetic and regulatory libraries for this organism make genetic circuits design and assembly challenging in this chassis.</p>
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<p>The ability to fine-tune gene expression forms a cornerstone for the design and operation of genetic circuits. This optimization has not been fully carried out in non-traditional chassis like L. lactis. Our project is aimed at the construction and testing of a library of synthetic 5’ untranslated regions (UTR) containing ribosome binding sites (RBS) in L. lactis. Creating a well characterized library of RBS sequences would enable the rational engineering of this bacterium towards increased yield of products such as hyaluronic acid (HA) and in biomedical applications like probiotics.</p>
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<p>The group led by Howard M. Salis at the Pennsylvania State University has designed a web tool, called the RBS Calculator, for the design of RBS sequences. While this tool has been immensely useful for synthetic biologists, the creators, and others, have highlighted the model’s drawbacks. In addition to our lab work, we also plan to study these drawbacks and come up with potential improvements to the existing model. This would include studying the effect of temperature on mRNA folding, the interaction between the mRNA and the ribosomal S1 protein, the difference in optimal RBS-AUG spacing in Gram-positive and Gram-negative bacteria, and the effect of RNAse-mediated degradation of the transcript. Combining the (hopefully improved) model with an optimisation algorithm, we aim to rationally design the ‘best’ RBS for a given gene, in a given organism.</p>
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<h1>PROJECT <br>DESCRIPTION</h1>
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Synthetic biology envisions a bioengineering domain for designing new genetic parts and systems, or redesigning of existing ones. Until now, the most versatile workhorse of synthetic biology has been Escherichia coli. However, there is a need for exploring new chassis which can be naturally adapted to unique traits or metabolic pathways.
Lactococcus lactis (L. lactis) is a ‘Generally Regarded As Safe’ (GRAS) organism. It is naturally found in milk products and is also known to colonize the human gut. Unlike E. coli, it does not have an endotoxin layer, which requires extra measures for purification. Due to its ability to use a wide range of substrates and tolerance for a wide range of conditions (pH, temperature, solvent concentration), L. lactis serves as an alternate bacterial model for metabolic and bioprocess engineering.
The range of products it can be engineered to produce include bioplastics, biofuels, biopolymers, polyols, and food flavors.
However, the absence of genetic and regulatory libraries for this organism make genetic circuits design and assembly challenging in this chassis.
The ability to fine-tune gene expression forms a cornerstone for the design and operation of genetic circuits. This optimization has not been fully carried out in non-traditional chasses like L. lactis.
Our project is aimed at the construction and testing of a library of synthetic 5’ untranslated regions (UTR) containing ribosome binding sites (RBS) in L. lactis.
In addition to our lab work, we also plan to study these drawbacks and come up with potential improvements to the existing model. This would include studying the effect of temperature on mRNA folding, the interaction between the mRNA and the ribosomal S1 protein, the difference in optimal RBS-AUG spacing in Gram-positive and Gram-negative bacteria, and the effect of RNAse-mediated degradation of the transcript. Combining the model with an optimisation algorithm, we aim to rationally design the ‘best’ RBS for a given gene, in a given organism.
