BACKGROUND: The sequencing of a newly discovered, unusual microorganism, Obscura
ID: 83442 • Letter: B
Question
BACKGROUND: The sequencing of a newly discovered, unusual microorganism, Obscura marylandium, reveals that it contains no membrane-bound nutrient receptors/transporters, which are commonly found in organisms ranging from bacteria, plants, and insects to mammals. Instead, it appears that metabolites needed by Obscura marylandium freely diffuse into (and out of) cells through unregulated pores. However, there are a number of small-molecule recognition proteins within the microorganism that bind tightly to metabolites. For example, research in your advisor's laboratory suggests that families of allosteric repressors sense intracellular nutrients and control the transcription of biosynthetic operons according to their quality and availability. Generally, it appears that the repressors can sense both precursors and end products of metabolic pathways. When end products are high in concentration, they stabilize monomeric forms of the repressor, which are able to bind DNA and inhibit transcription of biosynthetic operons. Alternatively, when precursors bind to the repressors, a shift to dimeric structures is promoted. Dimeric repressors are unable to bind DNA, and hence, transcription of biosynthetic enzymes is activated. Also, the lab has shown that there is an additional level of regulation: transcriptionally active repressor monomers bind to and activate a key enzyme that can covalently modify biosynthetic enzymes to increase their activity. Witht this background, please address the following questions:
1a) Without modifying the operator sites of biosynthetic operons, describe a synthetic biology design (or modification of the regulatory circuit described above) that would enable production of end products regardless of metabolite levels, effectively uncoupling metabolite levels from metabolite synthesis (assume any growth media is not nutrient limited). How would you test your design?
1b) In an alternative approach, how might you instill a bistable condition on the biosynthetic operons of Obscura marylandium so that they are either transcriptionally fully on or off, and are not operating at some intermediate level that is dependent upon metabolite levels?
1c) You discover that the repressors of many biosynthetic operons have fairly similar sequences, and postulate at your lab meeting that they derived from a common precursor protein involved in fatty acid metabolism. Your advisor urges you to demonstrate this hypothesis experimentally. What is your design? Please be specific regarding your approach and explain how you would verify your design.
1d) You are having a tough time transforming this microorganism with your plasmids. You recall from high school biology that some bacteria are naturally competent, in that they can take up DNA from the environment. Being a synthetic biologist, you realize this would be a useful attribute to enable your experiments on this organism. You scan the genome and find to your surprise that Obscura marylandium contains a complete set of ten genes that enable natural competence, but they are dispersed throughout chromosome and it is unclear how they are controlled. You decide it would be useful to design a genetic program to regulate these genes and establish a ‘natural competence circuit' to enable facile transformation. Describe your strategy to introduce a coherent control system for these genes that regulates their expression to maintain equivalent levels of their products for optimal uptake of DNA.
Explanation / Answer
1a) In these cases algorithms that rely on a catalog of known enzymes can be used to identify the shortest or most economical routes for the end product production. These heterologous enzymes can be installed within the working strain to enable production of the end product. Such de novo biosynthetic pathways will enabled biological production of end product compounds.
Multiplexed automated genome engineering (MAGE) will be used for mutations to microbial genes and genomes in a targeted fashion, Oligonucleotide-mediated genome editing will be executed, makes use of the phage lambda beta protein to integrate oligonucleotides bearing desired changes in the place of Okazaki fragments during DNA replication. such multiplexed modification of genomes allows the millions of biosynthetic pathway variants generated by in silico metabolic methods to be constructed in vivo. that would enable production of end products regardless of metabolite levels.
1b) The bistable structure is widely used as it provide two stable states for the cells and has a fixation effect on the stable state, and it is therefore feasible for experimental observation. Bistability was introduced into the genetic design of the synthetic operon to produce two different stable states which were design for two different biological functions essential for cell growth. A promoter would accelerate the expression of any of the two expression units occasionally showing slightly higher expression level, while the mutual inhibitory structure would suppress expression of the other unit, leading to a fixation effect of the expressed unit. Once genes involved in physiological functions responsive to external changes are introduced into the two expression units. The selective expression of the two units in cells can be considered a synthetic operon contributing to survival and/or adaptation.
1c) The FasR is a transcriptional regulator from Mycobacterium sp. that controls the expression of the fatty acid synthase (fas) and the 4-phosphopantetheinyl transferase (acpS) encoding genes and the products produced are involved in the fatty acid and mycolic acid biosynthesis pathways. We will demonstrate that fas and acpS genes are part of the same transcriptional unit and FasR specifically binds to three conserved operator sequences present in the fas-acpS promoter region (Pfas). The construction and further characterization of a fasR conditional mutant confirmed that FasR is a transcriptional activator of the fas-acpS operon and that this protein is essential for mycobacteria viability. Moreover, the combined used of Pfas-lacZ fusions in different fasR backgrounds will be determined by electrophoretic mobility shift assays experiments that will suggest that long-chain acyl-CoAs are the effector molecules that modulate the affinity of FasR for its DNA binding sequences and for the expression of the essential fas-acpS operon.
1d) Natural competence for transformation is a method adapted for a horizontal gene transfer in bacteria and contributes to the maintenance and evolution of bacterial genomes.Competence gene expression at the single cell level can be determined by providing evidence that under homogeneous conditions the majority of the cells express competence genes. Molecules contribute to competence induction will be based on carbon catabolite repression and quorum-sensing pathways, respectively. The contribution of these two signaling pathways to natural competence can be explored through natural transformation assays, transcriptional reporter fusions, quantitative RT–PCR, and immunological detection of protein levels using Western blot analysis. Thus we can suggest that tested competence genes are dependent on the transformation regulator TfoX.
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