Biotechnology: Principles and Processes Flashcards

Biology Chap 11

11714887093	Biotechnology	The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services.	0
11714956511	Genetic Engineering	Techniques to alter the chemistry of genetic material to introduce these into host organisms and thus change the phenotype of host organisms.	1
11715103985	2 Principles of biotech	Genetic engineering Maintenance of sterile ambiance to enable growth of only the desired microbe in large quantities to help in manufacture of biotech products.	2
11715121605	Characteristic feature of asexual and sexual reproduction	Sexual- permits variation by unique combinations of genetic setup Asexual- Preserve genetic info	3
11715130286	2 genetic engineering techniques that involve creation of recombinant DNA.	Gene cloning Gene transfer	4
11715133442	Why is genetic engineering preferred over traditional hybridisation?	Involves inclusion and multiplication of undesirable genes too Genetic engineering techniques introduce only 1 set of desirable genes in target organism.	5
11715145141	What prob occurs if an alien DNA enters our body? How can this prob be rectified?	Not be able to multiply itself in progeny cells of organism. Can be rectified if alien DNA is part of a chromosome of host called origin of replication and thus, replicated with it.	6
11715850713	Cloning	Making multiple identical copies of template DNA	7
11715913091	First instance of construction of an artificial recombinant DNA molecule	Linking a gene encoding antibiotic resistance with a native plasmid of Salmonella Typhimurium	8
11715917755	Plasmid	Autonomously replicating circular extra-chromosomal DNA Eg- pBR322 cloning vector	9
11715926496	Scientists who formed the first recombinant DNA	Stanley Cohen Herbert Boyer 1972	10
11715938459	Molecular scissors	Restriction enzymes Cutting of DNA at specific locations	11
11715957324	Function of DNA ligase in genetic modification	Acts on cut DNA molecules and joins their ends Eg- Linking of antibiotic resistance gene with plasmid vector	12
11715948573	Function of plasmid DNA	Act as vectors to transfer the piece of DNA attached to it.	13
11715969540	Recombinant DNA	New combination of circular autonomously replicating DNA created in vitro after genetic modification	14
11715982897	Basic steps of genetic modification of an organism	Identification of DNA with desirable gene Introduction of identified DNA into host Maintenance of introduced DNA in host Transfer of DNA to its progeny	15
11716012040	2 enzymes responsible for restricting growth of Bacteriophages in EColi.	1963 Milestone of biotechnology Methyl transferase- added methyl grps to DNA to protect it from fragmentation Restriction endonucleases- Fragmentation of DNA	16
11716034133	1st restriction endonuclease isolated	1968 Hind II Cut DNA molecule at particular point by recognizing a specific 6 b.p. sequence.	17
11716044313	Recognition sequence	Specific base pair sequence at which a restriction enzyme cuts the DNA	18
11716053554	Naming of restriction enzyme	1st letter- genus 2nd n 3rd letter- species of prokaryotic cell frm which they were isolated 4th letter- name of strain Roman no. after name- order of isolation of enzymes frm the strain of bacteria.	19
11716070404	Naming of Hind II	H- haemophilus in- influenzae d- d-type II- 2nd isolated RE	20
11716076443	Isolation of EcoRI	Escherichia coli RY 13	21
11716087518	Exonucleases	Remove nucleotides from the ends of DNA	22
11716088752	Endonucleases	Make cuts at specific positions within the DNA	23
11716096235	How does a restriction endonuclease function?	Inspects length of DNA sequence Finds specific recognition sequence Bind to DNA and cut each strand of double helix at specific points	24
11716124204	Palindromic nucleotide sequences	Palindrome in DNA is a sequence of base pairs that reads the same on 2 strands when orientation of reading is kept the same. Eg- 3'-->5' orientation	25
11716139778	At what point do REs cut DNA?	Cut the strand of DNA a little away from the centre of palindrome sites , but b/w the same 2 bases on the opposite strands	26
11716153453	Sticky ends	Overhanging stretches on each strand after action of REs.	27
11716157426	Why are sticky ends called so?	Form H-bonds with their complementary cut counterparts	28
11716161411	Use of sticky ends	Facilitates action of enzyme DNA ligase	29
11716177524	Use of REs	In genetic engineering to form recombinant molecules of DNA.	30
11716191312	Why should vector and foreign DNA be cut by same RE?	Same kind of sticky ends on both Joined 2gether end to end using DNA ligase.	31
11716211207	How are DNA fragments separated?	By gel electrophoresis	32
11716213927	Gel Electrophoresis	Technique used to separate biomolecules under electric field based on their size	33
11716223698	Principle behind gel electrophoresis	DNA fragments- -vely charged Forcing them to move to anode under electric field thru matrix Smaller fragment, farther it moves	34
11716238429	Commonly used matrix	Agarose gel Natural polymer extracted frm sea weeds Its sieving effect causes separation of fragments.	35
11716407242	Stain used in gel electrophoresis	Ethidium bromide	36
11716417101	How to see DNA fragments in visible light?	Staining of DNA Exposure to UV radiations Bright orange coloured bands seen	37
11716423733	Elution	Separated bands of DNA are cut out from agarose gel and extracted frm gel piece	38
11716442012	Why does an alien DNA need a vector?	Multiplication of alien DNA numbers to equal copy no. of plasmid or bacteriophage. Help easy linkage of foreign DNA Easy selection of recombinants frm non- recombinants.	39
11716490608	Features required to facilitate cloning in a vector	Origin of replication that supports high copy no. Selectable marker Cloning sites	40
11716501820	Origin of replication	Specific DNA sequence responsible for initiating replication	41
11716511440	Uses of ori	Piece of DNA linked to this sequence can replicate within host cells Controls copy no. of linked DNA	42
11716537694	Selectable markers	A gene introduced into a vector, that confers a trait suitable for artificial selection.	43
11716611088	Use of selectable marker	Identifying and eliminating non-transformants Selectively permitting the growth of transformants	44
11716618114	Transformation	Process by which piece of DNA is introduced in host bacterium.	45
11716633954	Why shld there be few or preferably 1 recognition site in vectors?	More than 1 recognition sites in vector will generate several fragments Complicates gene cloning.	46
11716652196	Where is ligation of alien DNA carried out?	Restriction site present in one of 2 antibiotic resistance genes.	47
11716676922	Selection of recombinants based on antibiotic resistance	Recombinants selected based on antibiotic resistant nature Non- recombinants- Resistant to ampicillin, tetracyclin Recombinants- Resistant to ampicillin	48
11716704891	How to isolate recombinants based on antibiotic resistance?	Plating of recombinants and non- recombinants in ampicillin and tetracyclin containing medium. Ampicillin- Both survive Tetracycline- Recombinants can't survive	49
11716799328	Why is insertional inactivation a better method of selection of recombinants?	Selection by antibiotic resistance is a time-consuming process as it req. simultaneous plating on 2 plates having diff. antibiotics.	50
11716904126	Insertional inactivation	Easy method of isolation of recombinants by colour production	51
11716973179	Principle behind insertional inactivation	Usage of selectable markers which can differentiate recombinants and non- recombinats on basis of ability to produce color in the presence of chromogenic substrate.	52
11716922255	Procedure of insertional inactivation	Insert incorporated in coding sequence of beta- galactosidase gene Recombinants- colorless due to inactivation of beta-galactosidase gene Non- recombinants- produce blue color in presence of chromogenic substance	53
11717013232	Why is insertional inactivation called so?	Insertion of foreign gene inactivates the enzyme	54
11717042226	Agrobacterium tumifaciens	Soil bacterium Pathogen of dicot plants Uses T-DNA to transform normal plant cells into tumour	55
11717090749	Ti plasmid	Tumour inducing plasmid Used if host is a plant cell T-DNA present in this Tumour inducing property is inactivated and it is now used as a cloning vector	56
11717108051	Ti plasmid is called natural gene engineer	During infection, Ti plasmid induced and tumor formed Due to this specific property of altering gene.	57
11717120252	Retrovirus	Ability to transform normal cells into cancerous cells Disarmed and used to deliver desirable genes in animal cells.	58
11717326545	Why can't DNA pass thru cell membranes?	Hydrophilic molecule The bacterial cells must be made competent to take up DNA	59
11717336017	Heat shock	Treat bacteria with specific conc. of Ca Recombinant DNA forced in cells by putting cells and DNA on ice, 42' conditions and ice again.	60
11717358913	Why are bacterial cells treated with Ca in heat shock method?	Method of insertion of recombinant DNA in host Increases efficiency with which DNA enters bacteria thru pores in cell wall.	61
11717367010	Micro injection	Method of insertion of recombinant DNA in host Recombinant DNA directly injected into nucleus of animal cell	62
11717373297	Biolistics/ Gene Gun	Method of insertion of recombinant DNA in host Cells are bombarded with high velocity micro particles of gold or tungsten coated with DNA Suitable for plants	63
11717383424	Usage of disarmed pathogen vectors	Method of insertion of recombinant DNA in host Disarmed pathogen vectors infect the cell and transfer recombinant DNA in host	64
11717409747	Processes of Recombinant DNA Technology	Isolation of DNA Fragmentation of DNA by REs Isolation of desired gene fragment by gel electrophoresis Ligation of DNA fragment in vector Transfer of recombinant DNA in host Culturing host cells in a medium on large scale Extraction of desired product	65
11717439275	Isolation of bacterial DNA	Lysozyme	66
11717443023	Isolation of plant DNA	Cellulase	67
11717445868	Isolation of fungal DNA	Chitinase	68
11717452623	Removal of RNA	Treatment with ribonuclease	69
11717461304	Removal of Proteins	Treatment with proteases	70
11717464876	Removal of lipids	Treatment with lipases	71
11717470444	How to obtain purified DNA after isolation?	Addition of chilled ethanol leads to precipitation of purified DNA Collection of fine threads in suspension	72
11717486409	How are restriction enzyme digestions performed?	Incubating purified DNA molecules with restriction enzyme at optimal conditions req. for that specific enzyme Electrophoresis to chk progression of digestion. After cutting by REs, ligation gv rDNA	73
11717532144	Polymerase chain reaction (PCR)	Multiple copies of gene of interest is synthesised in vitro using 2 sets of primers and DNA polymerase enzyme.	74
11717550243	Primers	Small chemically synthesised oligonucleotides tht are complementary to regions of DNA.	75
11717561636	Use of DNA polymerase enzyme	Extends primers by use of nucleotides in reaction and genomic DNA as template.	76
11717568765	Thermostable DNA polymerase	-Helps in repeated amplification of DNA by repeated replication of DNA -Isolated frm Thermus aquaticus bacterium -Remains active during high temp. induced denaturation step of PCR.	77
11717596015	How to induce further cloning of recombinant DNA?	Amplified fragment used to ligate with vector.	78
11717611323	Why is there a need for recombinant DNA to be expressed?	In almost all recombinant technologies, ultimate is to produce proteins, which is not possible unless DNA gets assimilated in host cells	79
11717657651	Why is there a need for large scale production of recombinant DNA?	Large amount of target proteins needed.	80
11717659855	Recombinant protein	Protein encoding gene is expressed in a heterologous host.	81
11717674033	How to produce higher yield of desired protein?	Cont. culture system- Used medium drained frm 1 side and fresh medium enters frm other. Maintains cells in physiological active exponential phase. Produces larger biomass	82
11717688543	Req. for bioreactors	Small volume cultures cannot yield appreciable quantities of products Large volumes of culture can be processed (100-1000L)	83
11717713253	Bioreactors	Vessels in which raw materials are biologically converted into specific products, individual enzymes, etc. using microbial plant, animals or human cells	84
11717729081	Use of bioreactors	Provides optimum growth conditions (temp., pH, substrate, salts, vitamins, O2) to achieve product	85
11717775875	Stirred- tank reactor	cylindrical or curved base- facilitates mixing of contents Stirrer- facilitates even mixing and O2 availability thru bioreactor Air can be bubbled thru reactor Agitator system O2 delivery system Foam control system Temp. control system pH control system Sampling ports- small vol. of culture withdrawn periodically	86
11717804975	Downstream processing	Series of separation and purification processes that protein has to go thru b4 marketing Formulated with suitable preservatives. Strict quality control testing	87