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
-
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
genome editing
be able to discuss biological origins and biotech use of genome editing techniques:
.crispr-cas9
.TALENs
.ZFNs
Genome engineering
ZFN-Zinc-finger nucleases
(ZFNs) are targetable DNA cleavage reagents that have been adopted as gene-targeting tools.
ZFN-induced double-strand breaks are subject to cellular DNA repair processes that lead to both targeted mutagenesis and targeted gene replacement at remarkably high frequencies.
An issue that needs more attention is the extent to which available mechanisms of double-strand break repair limit the scope and utility of ZFN-initiated events.
genome engineering
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3176093/
Making directed genetic changes is often called “gene targeting.
Procedures for gene replacement in baker’s yeast, Saccharomyces cerevisiae, have been available for several decades (Scherer and Davis 1979; Rothstein 1983)
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
ZFNS are optimizable but it takes effort:
(ZFNs) can induce targeted genome modifications with high efficiency in cell types including Drosophila, C. elegans, plants, and humans. A bottleneck in the application of ZFN technology has been the generation of highly specific engineered zinc-finger arrays
https://www.sciencedirect.com/science/article/pii/S1097276508004619
describe OPEN (Oligomerized Pool ENgineering), a rapid creating multifinger arrays, which we show is more effective than the previously published modular assembly.
OPEN to construct 37 highly active ZFN pairs which induced targeted alterations with high efficiencies (1%–50%) at 11 different target sites located within three endogenous human genes
PEN provides an “open-source” method for rapidly engineering highly active zinc-finger arrays, thereby enabling broader practice, development, and application of ZFN technology for biological research and gene therapy.
TALENs
transcription activator-like effector nucleases
they are made by xanthonas bacteria and delivered by type 3 secretion system to host cells
TAL effectors provide specificity in DNA sequence recognition via variability in the Repeat Variable Diresidue (RVD) at positions 12 and 13 of a 33-34 amino acid sequence
Can be used in a fusion protein with FokI nuclease – similar to ZFN
Cas9 (CRISPR)
1987, when an unusual repetitive DNA sequence, which subsequently was defined as a CRISPR, was discovered in the Escherichia colI
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5847661/
clustered reg interspaced palandromic repeats (bacteria and archaea)
RNA-guided nucleases from CRISPR-Cas systems are currently regarded as the most reliable tools for genome editing and engineering.
they are located in intergenic regions. (ii) they contain multiple short direct repeats with very little sequence variation, (iii) the repeats are interspersed with nonconserved sequences, and (iv) a common leader sequence of several hundred base pairs is located on one side of the repeat cluster.
Process of CRISPR-Cas acquired immune system. (Top) Adaptation. The invading DNA is recognized by Cas proteins, fragmented and incorporated into the spacer region of CRISPR, and stored in the genome. Expression (bottom). Pre-crRNA is generated by transcription of the CRISPR region and is processed into smaller units of RNA, named crRNA. (Bottom) Interference. By taking advantage of the homology of the spacer sequence present in crRNA, foreign DNA is captured, and a complex with Cas protein having nuclease activity cleaves DNA.
https://www.neb.com/en-gb/tools-and-resources/feature-articles/crispr-cas9-and-targeted-genome-editing-a-new-era-in-molecular-biology
Design strategy
using CRISPR/CAS9
https://bmcplantbiol.biomedcentral.com/articles/10.1186/s12870-020-02385-5
describe applications of genome editing tech question
how has gene editing gone from to concept clinical practice?
Li, H., Yang, Y., Hong, W.et al.Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects.Sig Transduct Target Ther5,1 (2020). https://doi.org/10.1038/s41392-019-0089-y
immunotherapy:
cells are isolated from a patient to be treated, edited and then re-engrafted back to the patient.
Target cells must be able to survive in vitro and return to the target tissue after transplantation.
cancer research:
a study used a human lymphoblast cell line derived from chronic myeloid leukemia (CML) patients, and a custom-designed ZFN was applied to this cell line to deliver site-specific DSBs to the telomeric portion of the mixed lineage leukemia (MLL) gene breakpoint cluster region as well as to analyze chromosomal rearrangements associated with MLL leukemogenesis via DSB error repair.
Furthermore, the use of HER2-positive cell-penetrating peptide (CPP) conjugated to mammalian mTOR-specific ZFN made the mTOR locus nonfunctional and inhibited relevant cancer signaling pathways, providing insight into the design of novel molecular targeted therapeutics for breast cancer
talens vs ZFNs
milestone of TALENs was achieved when they were primarily applied to efficiently disrupt the endogenous genes NTF3 and CCR5 in human leukemia cells via the introduction of NHEJ- or HDR-induced modification into a coding sequence, demonstrating that TALENs could be designed for selective endogenous gene cleavage.73 Interestingly, when TALENs and ZFNs were compared abreast at two human loci (CCR5 and IL2RG), TALENs showed a significant reduction in cytotoxicity. Moreover, the CCR5-specific TALEN was able to distinguish between the CCR5 target locus and a highly similar site in CCR2 when compared with ZFN technology
crispr uses
Human induced pluripotent stem cells (iPSCs) and CRISPR/Cas9 technology can also be combined to generate a congenital heart disease model associated with GATA4 mutations in vitro to investigate the pathogenesis of this gene mutation.
need to define the 3 and discuss the differences as well as the practical applications
