Genome Engineering
The CRISPR-Cas9 system has been successfully harnessed for various genome editing applications, which have a wide range of implications in fields such as medicine, agriculture, bioenergy, and food security. CRISPR systems contain two components: a guide RNA (gRNA) and a CRISPR-associated (Cas) endonuclease.
We will test PAM of various Cas9 enzymes:
1. SpCas9
2. SpyMac
3. ScMac
4. Sc++Mac
and introduce them respectively into bacterial cells using electroporation, which will turn on the trigger of genetic circuit-GFP, which will represent as fluorescent green if successful.
I. What tasks must a cell perform to live?
Cells provide six main functions. They provide structure and support, facilitate growth through mitosis, allow passive and active transport, produce energy, create metabolic reactions and aid in reproduction.
Debopriya Bose
II.What bacterial innate immune mechanisms must be overcome to perform genome transplantation?
Innate immunity mechanisms described so far include systems that prevent phage adsorption, phage DNA entry or that digest phage nucleic acids. When everything else fails, abortive infection systems trigger the ‘suicide’ of the infected cell, preventing phage multiplication to the benefit of the bacterial population.
There are three bacterial immunity mechanisms that are subjected to programmed genetic variation: first, the modification of surface elements that serve as phage receptors, second, the diversification of restriction–modification (RM) systems that attack phage genomes, and third, the generation of an arsenal of antiphage sequences in CRISPR loci.
DavidBikard et al
III.What mammalian innate immune mechanisms must be overcome to efficiently install large DNA molecules in mammalian cells?
The major functions of the innate immune system include:
Recruiting immune cells to sites of infection through the production of chemical factors, including specialized chemical mediators called cytokines
Activation of the complement cascade to identify bacteria, activate cells, and promote clearance of antibody complexes or dead cells
Identification and removal of foreign substances present in organs, tissues, blood and lymph, by specialized white blood cells
Activation of the adaptive immune system through a process known as antigen presentation
Acting as a physical and chemical barrier to infectious agents; via physical measures like skin or tree bark and chemical measures like clotting factors in blood or sap from a tree, which are released following a contusion or other injury that breaks through the first-line physical barrier (not to be confused with a second-line physical or chemical barrier, such as the blood-brain barrier, which protects the extremely vital and highly sensitive nervous system from pathogens that have already gained access to the host's body).
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