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Lecture 20

Biology 2581B Lecture 20: Lecture 20 genetics 2581

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Biology 2581B
David R Smith

Lecture 20  Craig Venter created the first biosynthetic prokaryotic micro plasmid bacterium = world’s first synthetic organism  Synthesized a complete genome and transferred it to a host cell  Rebooted the host cell so that it was under the control of the introduced synthetic genome  Can introduce synthetic tools into diabetic people to provide “cure”  How can we take all these process that occur in the cell, put it in a computer so that we can manipulate it and make it in a way that it can function the way we want  Example: insulin o Introduce these synthetic devices that will sort of prevent diabetes (to a certain point)  Take the genetic code and translate it to a binary code in the computer → one of the starting points for synthetic biology o Not as simple as taking genes, converting to genetic codes and putting in the computer → recombinant DNA technology = biotechnology  But a biological organism has so many processes organized at a hierarchical level, so it is not as simple as taking these genes and converting them to binary code and putting it into computers  There are certain processes and the genes within the genome code for different regulations  The gene needs to react to the environment and there is also communication between small molecules and proteins o External organisms that cause responses  You have to somehow figure out a way to put all these levels of these processes together and input them into a computer  Synthetic biology is a way to connect all these regulatory, sensory, physical, etc elements in a reliable and predictable way o The term predictable is really important since if you want to put a few genes together, you want them to give a predictable outcome - you want genes that respond to the environment in a predictable fashion o Apply engineering principles to the design and alteration of natural systems or de novo construction of artificial biological devices and systems that exhibit predictable behaviours  Need to program the cell to function as a whole system  You need to take advantage of a hierarchical system that already exists in biological systems  We already know what the cells are composed of and what proteins, etc they have and how they are designed to react  There is also an organism to ecosystem hierarchy (reacting to the environmental stimulus)  Since the hierarchy already exists in synthetic biology, it makes our job easier  You can alter the existing natural systems and create an entirely new system  Think about it in the context of a pathway, cell, or system as a whole, not just the level of the gene  Connect various elements/layers (regulatory sensory, etc.) in a reliable, predictable way and program a cell to function as an autonomous system, in the highest efficiency with the smallest genome possible (original definition)  Earlier known as recombinant DNA technology or biotech  Construct a biological system (ex. A network or a pathway) using characterized genes and regulatory DNA sequences  GOAL: achieve the highest efficiency in the synthetic cell with minimal genome that a cell can function efficiently for the cell to be able to adapt to any type of circumstance  Method: replicate the following areas “layers” of focus in a synthetic cell, but need to understand how they function first  Regulation: necessary to produce enough protein/RNA in the cell to perform their function properly o Transcriptional, translational, post-translational, modifications, epigenetic o Need to transform the cellular information into technological codes that can be input into computer  Sensory Stimulus: has to sense the signal within and outside the cell in order to perform functions such as glycolysis and photosynthesis o Essentially: how the cell reacts to its environment (both the outside and the inside) o Stimuli are how the cell detects these signals and transmits those to the interior of the cell  Stimuli: chemical, light, force (ex: chemical, kinetic, potential, pressure)  Communication: cell-to-cell communication, protein interactions OR how the cell communicates with other cells and its environment o Layer we are at now in biotechnology – we can create parts and components that do regulation and sensing, but need to create communication between cells to create multicellular organisms o Could be done through small molecules, proteins, viruses  Physical: response to motility, growth (photosynthesis, glycolysis), transport o Most important aspect of synthetic cells  Epigenetic: level of regulation based on the the environmental stimulus that exists  Synthetic Biology: Apply engineering principles to the design and alteration of natural systems or de novo construction of artificial biological devices and systems that exhibit predictable behaviors (new definition)  A computer can actually perform all the functions that a cell can do  Trying to synthesize parts that can function as a system and an organ by itself through technology, with the minimal genetic sequences/materials (genome) required in an efficient & predictable way  Take all the naturally existing system/programming inside a cell & transfer it into the computer  By the end, the synthetic organism must be able to survive in an unique environment that the scientists impose  Synthetic biology can be used at all Hierarchy and Modular Organization  Connects fundamental parts together (proteins) to get a desired outcome (products at the end of a biochemical reaction)  Multiple biochemical reactions together  Network of modules/biochemical pathways together  Multiple cells together to form a network  Parts to modules to complex systems, adapt from other disciplines and implement in biology (analogy)  Proteins and genes are the 1 layer = physical layer  A bunch of parts form gates. If connected together in a reliable fashion biochemical reactions = gates  Can input repressors or inducers that act as gates and produce proteins which act as the output  Gates put together form modules (pathways made up of multiple biochemical reactions)  Pathways make a cell functional  If you want to connect all the computers together, you make a network  If you want to connect all the cells together, you form tissues/cultures  Start by synthesizing/selecting DNA sequences containing the genes that contains desirable protein product  The amount of DNA selected & the order of genes = based on the identity of the desirable protein product  The product should participate in a biochemical reaction that you know would give you the desirable product (see circuit)  Scale for DNA synthesis & assembly:  In the order of genes: 10^2—10^4 bp  A gene circuit’s size: 10^4—10^6  A minimal genome: 10^6—10^7 (a combination of more than one circuits that is required to function, no embellishments)  This process is easy for bacteria = create a traditional recombinant plasmid since the sequences can be fused and inserted  Difficult for eukaryotes especially mammals, need to understand specific functions in each part of the genome  Challenge: unsure if the circuits will function as predicted once fused together in the minimal genome  NOTE: a microplasma = 1.8 mb (megabase) Synthetic Biology Circuits  Sensing, processing, actuation  Sense what is happening in the cell (microRNA, mRNA, and proteins) in the form of a regulatory circuit  If this and that are present, proceed to the next step  If you get to the end, you will get a genome  If you get too much production, you can kill the protein  microRNA, mRNA and proteins can all act as regulatory circuits  The microRNA / mRNA / proteins (physical layers) detect a signal in the environment & react together (circuit)  The final or byproducts serve as stimulus to the targeted molecules, the level of these products affect regulation (regulatory)  The process of proteins reaction together to serve regulatory functions is similar to programming: A+ B → C  If your logigate functions according to the logic command given (A+B), the product yield would be called “activation” (C)  In synthetic biology, the product C must behave in a predictable manner → must control the gate & logic command  Mutations create unpredictability in systems, force them to react to the mutation in different ways 1. Sensing 2. Processing 3. Actuation  Scale for DNA synthesis and assembly  Driving force for more synthetic creations = dropping costs  Trying to create a gene circuit so you put them all together using recombinant DNA technology  Increasing complexity as you go up the hierarchy  Can put in all kinds of stuff (e.g., repressors and genes) but it doesn’t mean it’ll function The Design Cycle  Must conceptualize what your goals, inputs, outputs are – and then you can design it  Select parts and the computer will put it together for you  Then you can start modeling the system – and then you can construct the system  Can use restriction digest to get the system  Once you get the system, you can probe, test, and validate it 1. Conceptualization → identify the system goals, necessary inputs needed to create the designed outputs 2. Design → understand network topologies, kinetic parameters, parts selection (all can be done on computers) 3. Modelling → how do circuits interconnect? Understand network behaviours, robustness, sensitivity 4. Construction → assemble & integrate into a plant 5. Probing, testing and validation → alteration, library screening, directed evolution *1-3 = design ; 4-5 = fabrication* Tools for Design Cycle: 1. Engineering principles for design (simply the process of construction)  Reduce efforts of design cycle  Decoupling: separate / taking apart each level of the cellular component to understand their functions / structures (simplification – rip system apart and see what’s in it)  Abstraction: extract the components from the cell/host, identify how they fit together to produce a viable system (separation into hierarchical levels, see how the pieces fit together)  Standardization: manipulating the separated components in such a way that the components should be able to function properly aka when we put input, an output should come out = putting them back together 2. Components for parts selection  Parts are designed and cataloged onto online databases, then parts are used to build a circuit  Example: anything that is important to gene expression (cis-elements, promoters, exons, protein domains, ORFs, terminators, initiation sites)  Biobricks – things available for purchase  Phytobricks – plants  Challenges: difficult to make sure that transcriptional regulation functions properly and hard to guarantee the precise control of expression in synthetic circuits & time- consuming  Otherwise: could yield unpredictable consequences in a circuit, and doesn’t work the way you want to 3. Computational tools for design and modelling  Help to design network and put parts together  Component design & synthesis (design network))  Can be obtained from the Library of Parts  Composed of the 4 areas of focus (regulation, sensor, communication & physical aspect)  When designing components, need to make sure that these 4 components are satisfied  Topology and network design (put parts together)  Behaviour predictions and simulation (see whether the cell survives)  Registry of Standard Biological Parts – a database where you can get biological parts from  A database from iGEM (international Genetically Engineered Machine)  Has more than 200 000 biological parts that could be used in the designing cycle Synthetic Microplasma Genome  Prokaryotic cell that contains a completely synthetic genome  No matter what changes you introduce to the system, they have to be predictable  Take parts like mRNA, miRNA, etc that are present within the cell and join them through regulatory systems, meaning parts A and B are going to turn on in response to a stimulus and produce an output  In order to build these parts you need DNA  So if you were to create gene circuits then you would have to fuse a few genes and their regulatory elements for the genes - cis elements, promoters, operators, etc that will control the expression and repression of these genes  You would make a plasmid that you would insert  If you're creating an entire genome, you have to consider the amount of DNA that is required (from mb to gb)  You have to take these little parts and stitch them together in a way that they can have a predictable function  To be able to do that, you have to follow a design cycle  First, you want to conceptualize what your end product is supposed to do  Think about your goal, input and outputs  Then you can design your model to reflect the given input and produce a predictable output  Then you have to actually construct and stitch these
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