Molecular biology george Flashcards
synthetic biology: should know
definition in terms of engineering novel pathways and in terms of engineering synthethic life
importantt echnologies in underpinning synthetic biology
examples of synthetic biology that equates to biotec applications
the lac operon/how gene expression is induced
has an IPTG (reagent) a molecular mimic of allolactose thus an inducer that will bind with the repressor and induce transcription in lac operon so then the operator becomes functional and the lac’s become active.
synthetic biology
(genetic engineering)
takes well characterised genetic parts and re-assembles them to create a new whole organism/molecule
the repressilator
(in ecoli)
Elowitz M.B., Leibler S. (2000) A synthetic oscillatory network of transcriptional regulators Nature 403, 335–338 doi:10.1038/35002125
the design of a synthetic network to do a particular function.
Using 3 transcriptional repressor system not attached to any biological clock to create a oscillatory network
the network periodically induces the immitence of GFP as a readout of its apperance in cells.
this has to be imputted generation to generation due to its speed.
The clock showed random behvaiour due to fluctuations of its components.
outcome depends upon:
repressor concentration
transaltion rate
decay rates
genetic circuit designs
Principles of genetic circuit design (2014) Jennifer A N Brophy& Christopher A Voigt Nature Methods 11, 508–520 (2014)
doi:10.1038/nmeth.2926
Engineers need to harness this capability to program cells to perform tasks or build chemicals and materials that match the complexity seen in nature.
challanges of circuits:
-circuits require the precise balancing of their component regulators to generate the proper response
-screening for dynamic circuits, such as oscillators, is significantly more complex60 and it is hard to imagine how screens would be established for more sophisticated functions
how?
-Transcriptional circuits operate by affecting the flow of RNA polymerase (RNAP) on DNA.
-DNA-binding proteins can recruit or block RNAP to increase or decrease the flux, respectively
whats next?
-computational tools have to be developed to aid the design process. These programs need to be able to simulate the dynamics of a circuit and convert the designs into a linear assembly of genetic parts
-new approaches to whole cell omics measurements have to be integrated into the debugging cycle
- a test for circuits that are difficult to control
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biobricks?
example
the parts used in synthetic biology
e.chromi- the creation of different coloured biosensors.
enabiling bacteria to respond to imputs (imput sensitive promotor)
The synthesized artemisinic acid is transported out and retained on the outside of the engineered yeast,
synthethic biology first success
Production of the antimalarial drug precursor artemisinic acid in engineered yeast
using vost effective method: semi-synthesis of artemisinin
done by:
engineering of (brewers yeast) Saccharomyces cerevisiae to produce high titres (up to 100 mg l(-1)) of artemisinic acid using an engineered mevalonate pathway, amorphadiene synthase, and a novel cytochrome P450 monooxygenase (CYP71AV1) from
mevalonate pathway
sequence of cellular reactions leading to farnesyl pyrophosphate, the common substrate for the synthesis of cholesterol and for protein prenylation (a post-translational modification necessary for the targeting and function of many proteins).
Synthetic biology for biofuels
the synthesis of petrolium replica (hydrocarbons) fuels by targerted mods on free fatty acid pools in e.coli
how?
The activity of the fatty acid (FA) reductase complex from Photorhabdus luminescens was coupled with aldehyde decarbonylase from Nostoc punctiforme to use free FAs as substrates for alkane biosynthesis. This combination of genes enabled rational alterations to hydrocarbon chain length (Cn) and the production of branched alkanes through upstream genetic and exogenous manipulations of the FA pool. Genetic components for targeted manipulation of the FA pool included expression of a thioesterase from Cinnamomum camphora (camphor) to alter alkane Cn and expression of the branched-chain α-keto acid dehydrogenase complex and β-keto acyl-acyl carrier protein synthase III from Bacillus subtilis to synthesize branched (iso-) alkanes. Rather than simply reconstituting existing metabolic routes to alkane production found in nature, these results demonstrate the ability to design and implement artificial molecular pathways for the production of renewable, industrially relevant fuel molecules.
the reductionist approach?
francis crick “he ultimate aim of the modern movement in biology is to explain all biology in terms of physics and chemistry” epitomizes the reductionist”
Reductionists analyse a larger system by breaking it down into pieces and determining the connections between the parts. They assume that the isolated molecules and their structure have sufficient explanatory power to provide an understanding of the whole system
a minimal genome
mycoplasma genetallium (yeast)
https://www.science.org/doi/10.1126/science.aad6253?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%20%200pubmed
what?
In 2010, a 1079-kb genome based on the genome of Mycoplasma mycoides (JCV-syn1.0) was chemically synthesized and supported cell growth when transplanted into cytoplasm. Hutchison III et al. used a design, build, and test cycle to reduce this genome to 531 kb (473 genes). The resulting JCV-syn3.0 retains genes involved in key processes such as transcription and translation, but also contains 149 genes of unknown function.
CVI-syn3.0 is a working approximation of a minimal cellular genome, a compromise between small genome size and a workable growth rate for an experimental organism. It retains almost all the genes that are involved in the synthesis and processing of macromolecules. Unexpectedly, it also contains 149 genes with unknown biological functions, suggesting the presence of undiscovered functions that are essential for life. JCVI-syn3.0 is a versatile platform for investigating the core functions of life and for exploring whole-genome design.
classifcation of how essential genes are by transposon mutagensis
utilizes transposable genetic elements that integrate into a recipient genome to generate random insertion mutations which are easily identified.
khorana chemical synthesis
(total synthesis of a gene)
https://pubmed.ncbi.nlm.nih.gov/366749/
Har Gobind Khorana developed a chemical method that was very important in synthesizing the RNA molecules with defined combinations of bases. These defined combinations of bases are the homopolymers and the copolymers. He used the synthetic DNA to prepare the nucleotides with the known repeating sequences
ZFNS
the challenge in extending gene targeting to other organisms and situations could be viewed largely as one of increasing the frequency of recombination
function:
ZFNs are used to create different repair outcomes by causing a double strand break.
Chromosomal breaks are detected in cells as potentially lethal damage, and one natural pathway of DSB repair is copying from a homologous template. From this perspective, DSB-stimulated gene targeting simply provides an exogenous template for a natural repair process. An alternative repair pathway for DSBs, nonhomologous end joining, often joins the broken ends inaccurately, creating deletions, insertions, and substitutions at the break site. Thus, both mutagenesis and gene replacement are stimulated locally by DSBs
