MUNI SCI Bi4025en Molecular Biology Mgr. Jiří Kohoutek, Ph.D. 1 Department of Experimental Biology Lecture 12 • Basic principals of genetic engineering 2 Department of Experimental Biology MUNI SCI Genetic engineering - definition • Definition by International Union of Pure and Applied Chemistry (IUPAC): • Process of inserting new genetic information into existing cells in order to modify a specific organism for the purpose of changing its characteristics •Also Known as Recombinant DNAtechnology, gene modification, and gene therapy. • Genetic engineering is a term that was first introduced into our language in the 1970s to describe the emerging field of recombinant DNA technology. • The recombinant DNA technology started with pretty simple things - cloning very small pieces of DNA and growing them in bacteria. • In a broad view the genetic engineering, is more defined as an entire field of recombinant DNA technology, genomics, and genetics. 3 Department of Experimental Biology MUNI SCI Gene - Genetic engineering • Genetic engineering is targeted construction of cells/organisms carrying such gene and protein variants and a combination of genes that do not exist in the nature. • Methods to propagate new molecule of DNA in new variant of the protein: o Joining DNA fragments isolated from different species, genera, families, etc... o Creation of completely new DNA molecules. o Transfer of modified molecules into cells and organisms. o Direct mutational interventions in the genome of cells and organisms. 4 Department of Experimental Biology MUNI SCI Gene - Genetic engineering • DNA manipulation in vitro, DNA amplification, gene cloning and their targeted modifications, transfer to cells / organisms (heterologous expression). • Targeted changes in genetic information may also be carried out in vivo (genome editing, chromosome engineering). 5 Department of Experimental Biology MUNI SCI Key discoveries • Restriction endonucleases and other enzymes for DNA manipulation: o DNA cutting in a precisely defined location. o Ligation of two foreign DNA (originating from different organisms), o DNA synthesis in a test tube ("artificial bacteria"). • DNA sequencing: o Determination of the molecular structure of the gene and regulators sequences. • Gene cloning: o Use of the universal genetic code and the method of expression of genetic information, o Introduction of the gene into the unrelated organisms (overcoming interspecies barriers), o Gene amplification to the unlimited quantities. o Targeted introduction of mutations into the gene (function improvement), o Study of the manifestation of altered genes (mutation x function). 6 Department of Experimental Biology MUNI SCI Key discoveries • 1965 - Discovery of plasmids. • 1970 - isolation of the first restriction enzyme. • 1972 - Preparation of the first recombinant DNA molecules in vitro. • 1973 - the beginning of gene cloning. • 1975 - The Asilomar Conference on recombinant DNA. • 1977 - First recombinant DNA molecules carrying mammalian genes. • 1977 - DNA sequencing. • 1978 - preparation of human insulin in bacteria (since 1982 produced commercially). MUNI 7 Department of Experimental Biology r> r» t Key discoveries • 1980 - First attempts of gene therapy. • 1983 - Discovery and introduction of PCR, an alternative to gene cloning in vectors. • 1997 - animal cloning, Polly the sheep (injection of genomic DNA into the nucleus the human blood clotting factor, exogen present, but not expressed). • after 2000 - genome editing - recombinant DNA nucleases -meganucleases recognizing long target sequences, CRISPR/Cas. 8 Department of Experimental Biology MUNI SCI Use of genetic engineering • Basic Research: • Study of the structure and function of genes and genomes. • Elucidation of basic processes of gene expression - recombinant DNA, reporter genes, etc. • Characterization of regulatory mechanisms of gene expression. 9 Department of Experimental Biology MUNI SCI Use of genetic engineering • Applied Research: • Production of medical agriculture and industry relevant substances: o Introduction of foreign genes into unrelated organisms and obtaining products in large quantities - overcoming reproductive barriers. • Preparation of substances with new properties: o Modification of existing genes or creating new ones - enzymes, antibodies, vaccines, etc o Improving the properties of therapeutic substances. • Alteration and improvement of the characteristics of organisms: o Preparation of microorganisms for biotechnology (e.g. Enhanced production). o Increased yields of cultural plants and livestock performance (resistance to diseases, pests or external influences, production of foreign substances in the bodies of plants and animals). • Gene therapy - treatment of genetic diseases. 10 Department of Experimental Biology MUNI SCI Particular steps of genetic engineering • 1) Insertion of the gene int the Plasmid - Gene cloning. • 2) Transfer of vector in the given organism - bacteria, plant, yeast, eukaryotic cells, etc. By transformation/transfection, electroporation, transduction, infection, microinjection, shooting gene. • 3) Expanding of bacteria. • 4) Basic or Applied Research. Bacterium O Gene inserted Into plasmid Cef I containing gerte of interest Bacterial Plasmid chromosome V^_-^^~~^Gene < Recombinant^—^ ™. , , ji interest DNA [plasmid) 1 © Plasmid put into * bacterial cell UNA of chrom osome Recombinant bacterium Gene of interest Copies ol gene ^V' Basic research on gene © Host cell grown In culture, to form a clone of cells containing the "cloned" gene of interest > Protein expressed by gene of interest O Basic research and various applications Protein harvested Basic research on protein Gene for pest Gene u sed to alter p rote! n d Issd Ives Human g rowth h or- resistance inserted bacteria for cleaning blood clots in heart mone treats stunted into plants up toxic waste attack in erapy growth 11 Department of Experimental Biology http://frhonorsbiologybeno.weebly.eom/biotechnology.html# MUNI SCI Gene cloning - i 11 Fragment A (PCR-annnlil'tie a FteBlrictlon Enzymes -mcs- OH Vector A f Vector B 1 Enzymsa Restriction sites at the DNA and plasmids are digested by the corresponding restriction enzymes (RE). The cleaved DNA is then ligated to a plasmid vector possessing compatible ends. DNA fragments can also be moved from one vector into another by digesting with REs and ligating with compatible ends of the target vector. Assembled construct is then transformed into Escherichia coli (E. coli). ^ , , https://international.nebxom/tools-and-resources/feature-articles/foundations-of- MUNI 12 Department of Experimental Biology iii-'*'*^** * * |" r a' molecular-cloning-past-present-and-future Q P T Plasmid selection A plasmid typically contains an antibiotic resistance gene, which allows bacteria to survive in the presence of a specific antibiotic. Thus, bacteria that took up the plasmid can be selected on nutrient plates containing the antibiotic. Bacteria without a plasmid will die, while bacteria carrying a plasmid can live and reproduce. Each surviving bacterium will give rise to a small, dot-like group, or colony, of identical bacteria that all carry the same plasmid. 13 Department of Experimental Biology https://www.khanacademy.org/science/ap-biology/gene-expression-and-regulation/biotechnology/a/overview-dna-cloning MUNI SCI Production of cloned gene • Once the bacterial colony with the right plasmid is verified. • After specific induction the large culture of plasmid is then grown • The bacteria serve as miniature "factories" churning out large amounts of protein. Co\owy Ys ^toudv-\ 5 14 Department of Experimental Biology -> MUNI SCI Cloning of genomic library Genomic DNA library isolation of DNA from cells cell Ii cens op o- digestion with restriction enzymes \. ligation with >H DNA ligase ^ amplification of genomic DNA library collection of genomic DNA library © 2012 Encyclopaedia Britannica, Inc. 15 Define footer - presentation title / department A genomic DNA library is a collection of DNA fragments that make up the full-length genome of an organism. A genomic library is created by isolating DNA from cells and then amplifying it using DNA cloning technology. https://www.britannica.com/science/genomic-library MUNI SCI Mutagenesis in vitro • „Site - Directed Mutagenesis". • Aims: o Analysis of the relationship between structure and function of nucleic acids, o Clarification of gene function and regulatory regions, o Targeted changes in amino acids in proteins. o Controlled evolution (bioinformatics tools, domain modeling, gene preparation, cloning and function test) - protein preparation with new properties (protein engineering), o Preparation of transgenic organisms • Procedure: o DNA isolation - change in DNA in vitro - transfer to the cell - evaluation of the effect. • Types of mutations: substitution, deletion, insertions. 16 Department of Experimental Biology MUNI SCI Mutagenesis in vitro • Random • Handling of restriction points. • Chemical mutagenesis • Incorporation of erroneous bases. • Gene search, or functional areas on DNA. • Targeted • Oligonucleotides mutagenesis (placement of mutations in a specific gene site). • Cassete mutagenesis (gene synthesis). • Substitution of bases or codons targeted changes in the structure of proteins. 17 Department of Experimental Biology MUNI SCI Restriction enzyme mediated mutagenesis in vitro Overhanging ends are: removed by Si nuclease, synthesized by DNA-polymerase -also just limited synthesis by chosen nucleotides. Generation of deletions or insertions within the genome. DNA Polymerase 18 Department of Experimental Biology 4 bp iniefled MUNI SCI Oligonucleotide mediated mutagenesis Parental strand / Newly synthesized strand Mutagenic primer Heteroduplex DNA Parental homoduplex • E. Coli transformation Mutated homoduplex 19 Department of Experimental Biology MUNI SCI Oligonucleotide mediated mutagenesis CCACGAGA 1 T GGT A CTCT CCA T GAGA 20 Department of Experimental Biology J- Mutant Strand Syndesis Perf&mn thermal cycling td: * Denat-jie DNfl template + Anneal mutagenic primers containing desired mutaiion * Exiend and incorporate pfim&rs ftrfh high-fidelity DNA polymerase GGT A CTCT CCA C GAGA Dpn\ Digestion of Template Digest parental methylated and h&nimeiheated DMA with Dpn I Primer GGT A CTCT 3F Transformation Transform mutated molecule into competent cells, tor nsck repak https://www.agilent.com/en/quikchange-ii-site-directed-mutagenesis-kits-details MUNI SCI Cassette mutagenesis Generally nearly 100% of the mutant clones. The limitation is presence of unique, conveniently spaced restriction endonuclease recognition sites. Xbal PI asm id vector w L Bglll Cleave DNA with Xbal and Bglll Remove wild-type fragment Wild-type DNA fragment Xbal Two synthetic oligonucleotides 'Anneal complementary oligonucleotides Xbal RZdb Bglll Bglll DNA "Cassette* DNA ligase Transform competent bacterial cells Mutant DNA Prepare plasm id DNA All resulting plasmids contain mutant sequence „„ ^ ,_ , , http://what-when-how.com/molecular-bioloqy/site-directed-mutaqenesis-part-1- MUNI 21 Department of Experimental Bioloqy . " a' a r- f f molecular-biology/ Q P T In vivo mutagenesis - gene editing • Genome editing with engineered nucleases. • Since 2000. • Targeted changes to any gene in any organism in vivo. •Artificially prepared nuclease introduces insertions, deletions, or replacement of the existing sequence at selected location within the genome. • Nucleases generates double-stranded breaks at designated points in the genome, thereby stimulating natural repair mechanisms in cells based on: o Homology-directed recombination - HDR - HR o Non-homologous end-joining - NHEJ. 22 Department of Experimental Biology MUNI SCI In vivo mutagenesis - Endonucleases V -Mi • Meganucleases • Zinc-finger nucleases - ZFN • Transcription Activator-Like Effector Nuclease - TALEN Clustered Regulatory Interspaced Short Palindromic repeats -CRISPR Molecular scissors- Genome Editing Tools Meganuclease S'-ATTATCTTCCAGA' 3'-TAATAGAAGGTCTA TCAGCAACCTGACA-5' ■AGTC GTTGG ACTGT -3' ZFN :irl lanaří 5-TTCGATATTCAGCAAC CTGACA GAAGTGGCTAAGGCTC-3 3'- AAGCTATAAGTCG TALEN STTG^GAC ACTGT CTTCACGCATTCCGAG-5' UJ UJ ZF modules Fokl nuclease 1".......... 5'■ TÁtTČÁČPíďť^ÁáŮ^i^li^i GTTTCATAOCCCGCACAC AGGAAGCGATATGTGAGTCGA -3 3' -ATGAGTGTGAGTGTTC TAGA CAAAGTATCGGGCGTCTG TCC^3^CGC5AWCACTCAGCT-5- VVvwAvivvvvvvliQw CRISPR/Cas9 r CAACTCCCTAAOOCTCAMIAT U gRNA p,) AAQATCC .I 0 PAM ATTCCOAGTTC1AGG RVD modules Fokl nuclease gRNA Cas9 nuclease 23 Department of Experimental Biology https://academic.oup.com/pcp/article/56/3/389/2460802?login=false MUNI SCI In vivo mutagenesis - Repair Mechanism • Double strand break can be repaired by: • NHEJ - insertions, deletions. • HDR - integration of new sequences. Double strand break ZFN .................................... I II III III IIIIIIIMI Mill lllllllll III Zinc fingers Nuclease TALEN III i 11 11 11 11.....i.....i.....i......... 111 ' " 11111'1.......1111.........111 TALE Nuclease CRISPR/Cas9 Double strand repair NHEJ .......1 ......i i 11 i " ■'....... Insertion ...... ■■Illlllllllll ..........11 ■1 11111 111,1 Deletion OR i i 11— ■ ' ■ ■_ lllllllll HDR ........... ........... 11 1111 I I TT TTT1 i i i i ■ ■ ■ ■ Donor DNA temple ........■.....-1 I 11 11 I I 11 11 I I .............. '■".......... Precise modification Department of Experimental Biology https://www.nature.com/articles/s41392-019-0089-y MUNI SCI Meganucleases • There are present in archaebacteria, bacteria, yeast, fungi and plants. • Meganucleases identify approximately 14-40 base pairs in the target sequences. • 5 families of meganucleases are known. The most widespread of them "LAGLIDADG family". • Meganuclease I - See I - recognizes the 18 nucleotide sequence 'TAGGGATAACAGGGTAAT', cleaves 4 nucleotides and leaves an end with overlap. • Complications - the 18 nucleotide sequence recognized by the l-Scel meganuclease would hypothetically require a genome 20 times larger than the human genome for the sequence to occur randomly in the genome. • The off-target activities induced by meganucleases are affected by the structure of meganucleases, and the delivery methods. 25 Department of Experimental Biology MUNI SCI Meganucleases • Meganucelases form dimer at the target site. • Each monomer can form a|3pap(3a fold, with four-stranded antiparallel p-sheets to recognize and combine with target sequence. • l-Onul meganuclease - 290 residues and is bound to a 22 base pair DNA target site. • The N-and C-terminal domains of the endonuclease recognize and interact with the 5' and 3' half-sites of the DNA target site. • The interface between the target 5' half-site and the protein N-terminal domain is indicated by the oval. 26 Department of Experimental Biology Clin. Transl. Med.2020; 10:412-426. Nucleic Acids Res. 2018 Jun 1 ;46(10):4845-4871. MUNI SCI Zinc-finger nucleases - DNA binding domain Structure of C2H2 zinc finger (C, cysteine; H, histidine) • The C2H2 zinc finger protein (ZFP) is one of the most common DNA binding motifs in multicellular organisms. The zinc binding coordinated by the amino acids Cysteine or Histidine. • It is capable of binding DNA/RNA/Proteins. • Individual fingers contain about 30 amino acids and these units typically occur as tandem repeats of two or more fingers. • Fusing the nonspecific DNA cleavage domain of the Type II restriction enzyme, Fokl, to a ZFP allows the resulting zinc finger nuclease (ZFN) to cleave DNA at a sequence determined by the ZFP. 27 Department of Experimental Biology https://www.med.nagasaki-u.ac.jp/phrmch1/lcn/interspersed_NLS.ritm MUNI SCI Zinc-finger nucleases - DNA binding domain • Structure of the Zif268-DNA complex. • Fingers are spaced at 3-bp intervals. • The structure and primary DNA contacting residues of zinc finger 2 (ZF2) are indicated to the right blue rectangle. • A sequence alignment of the 3 fingers of Zif268. The zinc binding Cys2-His2 motif is indicated with blue bold font; the canonical DNA-contacting residues are indicated by arrows in the red rectangle. ZF1 ZF2 -12 3 ZFl ZF2 ZF3 \W 1 FSRSDELTRH MERPYACPVESCDRRFSRSDELTRHIRIHTGQK PFQCRI—CMRNFSRSDHLTTHIRTHTGEK PFACDI—CGRKFARSDERKRHTKIHLRQKD if 28 Department of Experimental Biology Nucleic Acids Res. 2018 Jun 1 ;46(10):4845-4871. MUNI SCI Zinc-finger nucleases - Fokl endonuclease • Fokl endonuclease occurs naturally in Flavobacterium okeanokoites. • Consists of an N-terminal DNA-binding domain distinguishing a 5' GGATG 3' binding site and a C-terminal domain capable of non-specific DNA cleavage. • After binding to double-helix DNA, a domain capable of cutting is activated, which subsequently cuts: o 9 nucleotides per 5' —► 3' string o 13 nucleotides per 3' —► 5' chain. 29 Department of Experimental Biology MUNI SCI Zinc-finger nucleases % % %i [g@@ |o|o|t| |o|a|c| • To generate a ZFP with specificity for the sequence GGGGGTGAC, three fingers are identified that each bind a component \ i / triplet. These fingers are then linked. G G G GG T G A C Cleavage Right ZFP Domain í\A ^ 11 I I I I I I I I I I \3 v\i V\3 v V5 >r Cleavage Left ZFP Domain 30 Department of Experimental Biology Nucleic Acids Res. 2018 Jun 1 ;46(10):4845-4871. • Sketch of a pair of zinc finger nuclease (ZFN) subunits bound to two halves of a DNA target. • Each ZFN contains the cleavage domain of Fokl linked to an array of three to six zinc fingers to specifically recognize sequences that flank the cleavage site. • The Fokl nuclease domains transiently dimerize across those central bases and cleave each DNA strand to generate a double strand break with 5 overhangs averaging 4 bases in length. MUNI SCI Transcription Activator-Like Effector Nuclease • TAL: Transcription Activator-Like. • A group of proteins capable of specific binding to DNA. • It is used by plant bacteria of the genus Xantomonas and affects the DNA of the guest/recipient. • RVD: Repeat-variable di-residues, these are amino acids found in the TAL at position 12 & 13 that determine the specificity to the target nucleotide. 31 Department of Experimental Biology Nucleic Acids Res. 2018 Jun 1; PthXol binding region :4845-4871. MUNI SCI Transcription Activator-Like Effector Nuclease • The histidine at position 12 in the repeat number 14 forms a hydrogen bond to the backbone carbonyl oxygen of residue 8 in the first a-helix, while the aspartate at position 13 forms a hydrogen bond to the extracyclic amino nitrogen of the cytosine base. 32 Department of Experimental Biology • Repeats 14, 15 and 16 interacting with the DNA, illustrating that consecutive RVDs (HD, NG and NN, respectively in these repeats) contact consecutive bases (in this case cytosine, thymine, and guanine) on the same DNA strand. MUNI SCI Nucleic Acids Res. 2018 Jun 1 ;46(10):4845-4871. Transcription Activator-Like Effector Nuclease • RVD: Repeat-variable di-residues are amino acids in position 12 and 13 determine specificity to the target nucleotide - T, C, A and G. Translocation 34aa repeat modules NLS AD NH2 _T TTATTCCCTOACC _I I 7 710MDA__ÍD- COOH RVD LTP EQWAI ASNGGGKQALETVQRLL PVLCQAHG NG = T HD = C NI = A NN = G or A LTP EG VVAIA SHHGG KQ AL ETVG R LL P V LC Q A H G Left TALE E A GTC C A G a t A G T C C a A t C T A TG A C a t *- aatta'tJt aít A C A T CiGiGiA G c C c[t'g]c C a a a A T C a G G t T A G a T AJC t G t a g ttaataa ta T G T a GCC T C G G G A CG G T T T Spacer (12-21 bp) \AAß Right TALE 33 Department of Experimental Biology MUNI SCI Clustered regularly interspaced short palindromic repeats V PňiliiH|iiin.T a PAM r-1 tracrE^A*-1 Ca*9 CaiLCasZ CsnJ Leader l íMcw spacer q integration Repeats I 1 In Pri-crRNA 5' crRNA v CKiSI'R array i 0 nrři^3 nj^" ij^™ i ¥ 5' .v J ■3' y — AdaptalionfA. B and C) — Expression (i), K and t) Inlirfcrijicc (G, K and I) (A) The host is invaded by phage DNA during infection. (B) The invading DNA is processed by various Cas genes into small DNA fragments (protospacer). The selection of protospacer depends in part on the specific recognition of protospacer adjacent motif (PAM) present within the viral genome. (C) The small DNA fragments is then incorporated into the CRISPR locus of the bacterial genome as a new spacer, flanked by a repeat sequence. (D) the CRISPR locus is transcribed into a long precursor CRISPR RNA (pre-crRNA). 34 Department of Experimental Biology Transgenic Res. 2021 Jun;30(3):221-238. MUNI SCI Clustered regularly interspaced short palindromic repeats V PňiliiH|iiin.T a PAM r-1 tracrE^A*-1 Ca*9 CaiLCasZ CsnJ Leader l íMcw spacer q integration Repeats I 1 In Pri-crRNA 5' crRNA v CKiSI'R array i 0 nrři^3 nj^" ij^™ i j> s' —1*3' s ■3' y — A Emmanuelle Charpentier: CRISPR Therapeutics 3536^5 B 171 nt- J J 1 89 nt i -75 nt ■ css1 )^^^| csn2 B63732 lepA ~)i ■ 511 nt ■ 66 nt — 39-42 nt tracrRNA, trans-activating CRISPR RNA 36 Department of Experimental Biology pre-crRNA 171/89 nt1 tracrRNA r ^^^Csn1 First processing event Second processing e-ven! 56 nt 66 nt 39-42 nt RNase I 66 nt 56 nt 39-42 nt MUNI SCI Structure of Cas9 Jennifer Doudna : Intellia Therapeutics & Caribou Biosciences SpyCa ■ Arü-rich region ňuvC domain ■ alpha-helical lobe ■ C-terminal domain Department of Experimental Biology Martin J, et al. 2014, Science, (6176):1215-1215; Wiedenheft B, et al., 2009, Structure, 17(6):904-912; Haurwitz R E, et al. 2010, Science, 329(5997): 1355-8. Preparation of dual tracrRNA-crRNA into gRNA A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity Martin Jinek,1'2* Krzysztof Chylinski,54* Ines Fonfara,4 Michael Hauer,2f lennifer A. Doudna.12rS'6± Emmanuelle CharDentier4± 17 AUGUST 2012 VOL 337 SCIENCE 38 Department of Experimental Biology Cas9 programmed by single chimeric RNA ilJIIIIIIIIIIIill hraei loop MUNI SCI CRISPR/Cas9 system H +n bp sgRNA offset .. 3' ■■ 5' Indel mutation codon Precise gene editing 5'-----—---3' 5'---—---3' 3'-----—---5' 3' -------5' 39 Department of Experimental Biology Ran et al. 2013. Nat Protoc. 8:2281 MUNI SCI Protein engineering Protein engineering is the conception and production of unnatural polypeptides, often through modification of amino acid sequences that are found in nature. Synthetic protein structures and functions can now be designed entirely on a computer or produced through directed evolution in the laboratory. Objective: to change the structure and function of proteins through recombinant DNA technology: o Changes in protein binding regions, o Thermostability. o Speed and substrate specificity of reactions, o Sensitivity to oxidation and toxic substances. Rational design Target protein Random mutagenesis or directed evolution Redesign ooo®oooo»o 00000*000» OOOOOOGOOO »ooooooiot ® o o o o o o o o c 00010**130 Redesign Screening Lirjand f V Li gaud V Llgand ■■■■<ä\cmNg£ Selection and analysis Target protein with desired characteristics Trends Biotechnol. 2020 Jul;38(7):729-744. MUNI 40 Department of Experimental Biology _ _ T https://www.nature.com/subjects/protein-engineering k Protein engineering - enzymes in washing powders • Enzymes in washing powders are strongly genetically modified in order to require traits achieved by genetic modification: o Water softening. o Detergent for removing impurities (proteins, dyes, polysaccharides, etc.). o Efficiency at high and low temperature, o Whitening effect. • Subtilisin - is a protease produced by Bacillus subtilis into the extracellular space, decomposes substrates and their products use as source of nutrients. Lysis a number of substrates. Modified to withstand high temperatures. Expansion for substrates spectrum. Resistance to bleach, o All changes were done by successive/step by step amino acid substitutions. o o o o 41 Department of Experimental Biology Felix Jakob, Master Thesis, "Engineering of subtilisin proteases for detergent applications", Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH, Aachen University zur Erlangung , 08.05.2013. MUNI SCI Protein engineering - Industrial application of eznymes Sector Enzymes Applications Pharmaceuticals Nitrile hydratase, transaminase, monoamine oxidase, lipase, penicillin acylase Synthesis of intermediates for production of active pharmaceutical ingredients Food Processing Trypsin, amylase, glucose isomerase, papain, pectinase Conversion of starch to glucose, production of high fructose corn syrup, production of prebiotics, debittering of fruit juice Detergent Protease, lipase, amylase, cellulase Stain removal, removal of fats and oils, color retention, Biofuels Lipase, cellulase, xylanase Production of fatty acid methyl esters, decomposition of lignocellulotic material for bioethanol production Paper and Pulp Lipase, cellulase, xylanase Removal of lignin for improved bleaching, improvement in fiber properties 42 Department of Experimental Biology Catalysts 2018, 8, 238; doi:10.3390/catal8060238 MUNI SCI Insulin • Insulin is produced by B cells of the islets of Langerhans of the pancreas and released in the blood. • Insulin lowers blood sugar levels (antagonizes glucagon function). • Used for the treatment of diabetes. • In humans composed of two polypeptides A and B connected by two disulfide bonds. 43 Department of Experimental Biology MUNI SCI Processing of insulin in mammals In mammals - preproinsulin (inactive as a hormone) is first translated from the insulin mRNA. Proteolytic processing is necessary to make biologically active insulin. (A) The linear protein preproinsulin contains a signal sequence, which is cleaved after the protein enters the ER, an A chain, a B chain, and a C-peptide. (B) Inside the ER, the proinsulin (insulin precursor) folds and disulfide bonds form between cysteines. (C) Finally, two cleavages release the C peptide, which leaves the A and B chains attached by the disulfide bonds. This is now active insulin. i—i s s I-1-1-, K-R-^l-V-E-q-OC-T-S+C-S-L-Y-Q-L-E-N-Y-C-N -- s s I s s ,-1-1-, I F-VN-Q-H-LCG-SHL-V-E-A-L-Y-L-V-t-G-E-R-G-f-F-Y-T-P-K-T I Department of Experimental Biology https://bio.libretexts.org/Bookshelves/Cell_and_Molecular_Biology/N-linked_Protein_Glycosylation_Begins_in_the_ER MUNI SCI Industrial production of recombinant insulin E coli O Recombinant DNA plasmid Lysate supernatant Induction Cell lysis and recovery of IBs Fermentation f _ , ,. , Pioinsulin Proinsulin fusion fusion protem Inclusion body (IB) containing proinsulin fusion prolein IBs pellet protein extraction CNBr cleavage, oxidative Buffer exchange lf, . . " sulfitolysis IBs (pellet) IBs solubilization I Refolding Proinsulin purification 45 Department of Experimental Biology Enzymatic cleavage Bioresour Bioprocess. 2021 ;8(1):65. Lysate pellet (IBs, cell debris, contaminant) V * Washed pellet (IBs) IBs washing * • Cell debris, -W contaminants Insulin purification Insulin polishing Formulation of purified insulin MUNI SCI Plant transformation by Agrobacterium tumefaciens Gene transfer from bacteria to plants occurs naturally. Agrobacterium tumefaciens is a soil pathogen, a gram-negative bacterium which infects many species of plants causing a disease known as "crown gall". It has two common species: o A. umefaciens o A. rhizogenes 46 Department of Experimental Biology https://www.thebiomics.com/notes/applied-biology/agrobacterium-tumefaciens-mediated-transformation.html MUNI SCI Plant transformation by Agrobacterium tumefaciens • The oncogenic activity of Agrobacterium is due to the presence of a large (200-kb) tumor inducing plasmid (pTi) and tumor formation results from its infection into the host plant (dicotyledonous plants). • Upon infection of the wounded plant, the bacterium transfers a small segment of DNA, the so-called T-DNAfrom the Ti plasmid into the plant cell. • The T-DNA is then translocated into the plant nucleus and stably integrated into the chromosome. • In the Ti-plasmid itself, the T-DNA is flanked by 25-bp imperfect direct repeats known as border sequences. 47 Department of Experimental Biology https://www.thebiomics.com/notes/applied-biology/agrobacterium-tumefaciens-mediated-transformation.html MUNI SCI Plant transformation by Agrobacterium tumefaciens • The wild type T-DNA encodes a specific set of oncogenic (one) genes that when it is expressed in the host cell, leads to the formation of a tumor. • T-DNA carries genes for phytohormone (auxin and cytokinin) and opines. • The overproduction of phytohormones at the site of infection is responsible for the proliferation of wound cells into a gall (tumor) that can harbor a population of bacteria. „„ ^ , , https://www.thebiomicsxom/notes/applied-bioloqy/aqrobacterium-tumefacien MUNI 48 Department of Experimental Biology "V . . . ' , .. ' . . . rr *} a _ _ _ |" r a' mediated-transformation.html Q P T Plant transformation by Agrobacteríum tumefaciens Agrobacterium tumefaciens bacterium (o_D Restriction cleavage site T-DNA O The plasmid is removed from the bacterium, and the T-DNA is cut by a restriction enzyme. O The plasmid is reinserted into a bacterium. Q Inserted T-DNA carrying foreign gene \© The foreign DNA is inserted into the T-DNA n of the plasmid. The bacterium is used to insert the T-DNA carrying the foreign gene into the chromosome of a plant cell. Recombinant Tt plasmid 0 The plant cells are grown in culture. O Foreign DNA is cut by the same enzyme. © A plant is generated from a cell clone. AN of its cells carry the foreign gene and may express it as a new trait. © BENJAMIN/CUMMINGS 49 Department of Experimental Biology https://sphweb.bumc.bu.edu/otlt/MPH-Modules/PH/GMOs/GMOs3.html MUNI SCI Plant transformation by Agrobacterium tumefaciens A B. : f * a » ZWAi 4WA • Schematic presentation of rice transformation. • GFP expression in callus 2 weeks after infection (A), 4 weeks after infection (B), 6 weeks after infection (C), and in regenerating shoots and roots (D). MUNI SCI 50 Department of Experimental Biology July 2013Plant Cell Tissue and Organ Culture 114(1). Use of genetic engineering in the plants • Food and feed. • Influencing agronomic properties: o Herbicide resistance. o Resistance to pathogens (insects, viruses, fungi, etc.). o Tolerance to stress (water stress - drought, temperature -frost; osmotic stress - salinization of soils). • Modification of post-harvest properties: o Extended shelf-life. o Slowing down maturation and inducing resistance to shelf storage diseases, o Refine nutritional value and taste. 51 Department of Experimental Biology MUNI SCI Use of genetic engineering in the plants • Production of secondary metabolites: o Pharmacological preparations. • Technical crops: o Production of starch and oils for industrial use. o Biodegradable plastics. • Phytoremediation: oUse of green plants to remove polluting substances from the environment. 52 Department of Experimental Biology MUNI SCI Transgenic plants Resistance to insect pests. The spores of bacterium Bacillus thuringiensis produce protein with toxic effects on insects Delta endotoxin = 5-endotoxin. ^ M . _ < Bacillus thuringiensis (Bt) 5-endotoxin is highly poisonous to some groups of insects, but completely harmless to mammals and humans. 5-endotoxin changes to a toxic form, the only in the intestine of insects. Previously bacteria Bacillus thuringiensis cultured in large volumes and as spraying applied to plants . Later the gene for 5-endotoxin transferred to plants directly -transgenic potatoes, corn, rice, tobacco, tomato, broccoli, cotton. Indirect way - cloning the gene for toxin into bacteria colonizing plants (leaves, roots)-e.g. Pseudomonas fluorescens. Department of Experimental Biology https://www.chemicalbook.com/ProductChemicalPropertiescb7848438_EN.htm Ri Transgenic BT-Maize • In addition, it contains 3 genes: • Gene for plant resistance to insect pests, o 5-endotoxin from B. thuringiensis. • Gene for resistance to the herbicide Basta. o Basta is a short-lived herbicide, that is environmentally friendly. o The gene for resistance comes from the bacterium Streptomyces. • Gene for resistance to the antibiotic ampicillin. o Selection marker used for selection of transgenic plants (cells). o The gene is of bacterial origin. Department of Experimental Biology Transgenic plants • Herbicide resistance: • Weeds negatively affect the yield and quality of the crop (competition for nutrients, light, pest source). • Herbicides are chemicals that disrupt certain metabolic processes in the plant and damage it. • Selective weed elimination is a problem, a number of herbicides are total - they also destroy cultural crops. • Only after the introduction of herbicide insensitivity transgenes into the genome of cultural crops is achieved its selectivity. 55 Department of Experimental Biology MUNI SCI Transgenic plants • Resistance to the herbicide glyphosate (Roundup). • Glyphosate - non-selective, total herbicide. • Effective on 76 of the 78 most important weeds. • Inhibits enzymes providing the synthesis of essential amino acids. • This enzyme is not present in animals - the herbicide does not harm them. • Strategy: o Introduction of a gene for the formation of the target enzyme into agricultural plants (a larger amount of enzyme will improve the resistance of plants). o Introduction of a gene for the formation of altered -more active enzyme. o Introduction of a gene for the formation of an enzyme that inactivates the herbicide. 56 Department of Experimental Biology Transgenic plants Improvement of nutritional values of fruits and seeds or plant products used industrially. FlavrSavr tomato from company Calqene. The tomato was made more resistant to rotting, by adding an antisense RNA which interferes with the production of the enzyme polygalacturonase. This enzyme is normally responsible for cell walls softening during the fruit ripening. m □ 3 f: - -■ □ Unmodified tomatoes are picked before fully ripened and artificially ripened using ethylene gas which acts as a plant hormone - easier handling and extended shelf-life. FlavrSavr tomatoes could be allowed to ripen on the vine, without compromising their shelf-life. 57 Department of Experimental Biology https://alchetron.com/Flavr-Savr https://www.bionity.com/en/encyclopedia/Flavr_Savr.html MUNI SCI Transgenic plants Improvement of nutritional values of fruits and seeds or plant products used industrially. Rice with provitamin A (carotene): Solves vitamin A deficiency in poor countries (consequence of night blindness, blindness). Three genes introduced into the rice genome, the expression of which leads to the formation of provitamin A. Further modification of rice aims to increase the iron content. Department of Experimental Biology Science. 2000 Jan 14;287(5451):303-5. PNAS, 2021, Vol. 118, No. 51, e2120901118 Wild-Type Transgenic K£___S H mi m MUNI SCI Transgenic plants • Improvement of nutritional values of fruits and seeds or plant products used industrially. • Brassica napus var. Napus, rapeseed - seed oil containing an increased proportion of acid. Laurie (soaps and detergents). • Rapeseed - seed oil rich in myristate (cosmetics), or erucic acid (lubricants and nylon production). • Arabidopsis and rapeseed - formation of biodegradable polymers in chloroplasts usable as plastics (polyhydroxybutyrate, polyester-like polymers in cotton fibers). • Decaffeinated coffee plant - due to spontaneous mutations a rare variant found in which a key enzyme for caffeine formation is absent. 59 Department of Experimental Biology MUNI SCI Transgenic plants - vaccine production of HBV antigen(s) parenteral vaccines by injection in a plant bio-reactor, e.g. tobacco Stable or transient Powdered lyophiiized tissue Oral formulation production of HBV antigen(s) as a semi-finished product e.g. tablet or capsule in an edible plant, e.g. lettuce for oral vaccine forms as a booster vaccine • Plants are suitable for passive immunization (eating transgenic plants containing fragments of antibodies) and active immunization (antibody induction). • Immunization by eating raw vegetables containing antigen (vaccine), which induces the production of immunoglobulins mucosal immune system in the digestive tract. o Hepatitis B virus surface antigen, o Subunit B toxin cholera. 60 Department of Experimental Biology Int. J. Mol. Sei. 2013, 14(1), 1978-1998; MUNI SCI Transgenic plants Developmental status of edible vaccines in clinical trials. Pathogen Antigen Host Use Clinical trial status Enterotoxigenic LT- B Potato Diarrhoea Early phase 1 E. coli Enterotoxigenic LT- B Maize Diarrhoea Early phase 1 E. coli Norwalk Virus CP Potato Diarrhoea Early phase 1 Rabies Virus GP/NP Spinach Rabies Early phase 1 HBV HBsAg Lettuce Hepatitis B Early phase 1 HBV HBsAg Potato Hepatitis B Phase 1 Vibrio cholerae CTB Rice Cholera Phase 1 HBV HBV Saccharomyces cerevisiae Chronic HBV Phase 2 61 Department of Experimental Biology Mol Biotechnol. 2020; 62(2): 79-90. MUNI SCI Transgenic animals • Instrumental tool to: • Study of the functioning of genes in the context of the whole organism. • Improving the performance characteristics of livestock. • Formation of foreign proteins. • Models of study of genetic diseases. • Searching for possibilities for gene therapy of human diseases. MUNI 62 Department of Experimental Biology r> r» t Transgenic animals - mouse Mus musculus - mouse Reproduction: o 5-10 litters / year o 5-10 pups / litter o 19-21 day gestation o Sexually mature at 7 weeks o 4-5 generations per year 63 Department of Experimental Biology Oncotarget, February 2019 10(12). MUNI SCI Transgenic animals - transgenic mouse 64 Department of Experimental Biology CUcr layer O Inner cell mass MioD-psJDttc [ES ce sj * . Sperr fifillflivsifin Blastocyst Introduce gene in:o Eü cells — Cutture ES cells Selort ( containing gene Inject transformed £>^>& ES cells back into blastocyst Transplant into surrogate Birth Mouse heterozygous for Inserted gene Normal mice j\ Transgenic mouse honnozygous for inserted gene Sotrea: Koniad Hahop. BSE Inqub-y, London. 2000 L B-ocd MUNI SCI Transgenic mouse - gene of interest insertion Microinjection of cloned gene into nucleus of newly fertilized egg. Transfection incubate ES cells in solution that makes them take up the DNA, very inefficient need to identify cells that took up the DNA with reporter such as drug resistance. Electroporation - a high voltage pulse "pushes" DNA into cells. Inner cell Trophoblast 1 mhryo donor parent mice Culture of embryonic stem cells Cloned gene in vector Mix embryonic stem cells with cloned gene • Retroviruses - a more natural way or getting genes into cells. 65 Department of Experimental Biology MUNI SCI Transgenic mouse - gene of interest insertion Method used in producing transgenic mice by the microinjection of exogenous DNA into the pronuclei of fertilized eggs. cf Fertile Transgene Collection of One-Cell Embryos Implant Injected Embryos into Pseudopregnant females Live Birth ^ Test for Transgene W Injection of Transgene into Male Pronucleus Department of Experimental Biology Transgenic Founder Animal ILAR Journal, National Research Council, Institute of Laboratory Animal Resource, February 1997, 38(3):125-136. Hi U II SCI Transgenic mouse - gene of interest insertion 1. Donor Blastocyst ES Cells Transqenic s.M.croinject m Transformed Cells Into Blastocyst 7. Implant/ 6. Recipient Blastocyst www.shutterstock.com ■ 1959156628 • Embryonic stem cells are cultured and subjected to DNA-mediated gene transfer or other manipulations. • Cells with the desired genetic alteration are then inserted into blastocyst cavities, whereupon they resume normal development and produce a genetically mosaic mice, with some cells derived from the ES line, and the others from the injected blastocyst. • Founders, usually males, are then bred to pass on the gene, provided the ES cells differentiate into sperm. The F1 hybrid then carries the new gene in all cells. „ _ rr. , , https://www.shutterstock.com/imaqe-vector/illustration-qenetic-enqineerinq MUNI 67 Department of Experimental Bioloqy r ... H^r^r^oo aaaa ^ ^ -■ r r a* mouse-production-1959156628 Q P T Transgenic mouse - gene of interest insertion zygote ESC-transfection drug ESCs selection (+/+) (-/-) 68 Department of Experimental Biology Sei Rep. 2016 Aug 17;6:31666. (+/-) monoallelic mutation (-/-) biallelic mutation mosaic (+/+, -/-) chimera MUNI SCI Transgenic mouse - gene of interest insertion Chimeric mice generated from ESCs carrying a biallelic mutation in Cetnl gene (em51/em52). Highly chimeric animals have darker coat color. Testes of B6D2F1 (wt) and chimeric mice (chi) were photographed under bright and fluorescent field. GFP Hoechst 10 pm 69 Department of Experimental Biology Sci Rep. 2016 Aug 17;6:31666. MUNI SCI Transgenic salmon AquAdvantage Salmon has been genetically engineered. It contains an rDNA construct that is composed of the growth hormone gene from Chinook salmon under the control of a promoter from ocean pout. W GM salmon r* Length: 24ins Weight: 6.61b Farm salmon Length: 13ins Weight: 2.81b V *Both fish are 18 months https://www.fda. gov/animal-veterinary/aquadvantage-salmon/qa-fdas-approval-aquadvantage-salmon Department of Experimental Biology https://www.soilassociation.org/blogs/2017/august/theres-something-fishy-about- genetically-engineered-salmon/ Transgenic animals Protein Source Against Antithrombin 111 Goal Thrombosis Tissue plasminogen activator Sheep, pig Thrombosis a-anlitrypsin Sheep Emphysema Factor VIII. IX Sheep, pig. cow Hemophilia ä -Glucosidase Rabbit Pompe's disease Fibrinogen Cow. sheep Wound healing Glularmic acid decarboxylase Goat Type 1 diabetes Human scrum albumin Cow. sheep Maintenance of bkxxl volume Human protein c Goat Thrombosis Monoclonal antibodies Chicken, cow. goal Vaccine production Pro 542 Goat HIV Lactoferrin Cow Gl tract infection and infectious arthritis 71 Department of Experimental Biology Agricultural Reviews, January 2015, 36(1). MUNI SCI Cloning of animals • Cloning is a controversial process that involves creating a genetically identical organism, or clone, from the genetic material of an individual. • Clones are created by inserting a nucleus from an individual into an unfertilized egg. The egg will develop as if it had been fertilized to create a new living organism. • In 1996, scientists were able to successfully clone the first mammal, a sheep, after 276 attempts. • The successful clone was named Dolly. Since Dolly, scientists have successfully cloned other animals, such as, cats, cows, dogs, and rabbits. Ian Wilmut University of Edinburgh 72 Department of Experimental Biology http://frhonorsbiologybeno.weebly.eom/biotechnology.html# MUNI SCI Cloning of animals Cloned Foster Lamb Mother 73 Department of Experimental Biology MUNI SCI Gene therapy • Gene therapy is a technique that modifies a person's genes to treat or cure disease. Gene therapies can work by several mechanisms: o Replacing a disease-causing gene with a healthy copy of the gene. o Inactivating a disease-causing gene that is not functioning properly. o Introducing a new or modified gene into the body to help treat a disease. • Gene therapy products are being studied to treat diseases including cancer, genetic diseases, and infectious diseases. MUNI 74 Department of Experimental Biology https://www.fda.gov/vaccines-blood-biologics/cellular-gene-therapy-products/what-gene-therapy O 0 J. Road Map of Gene therapy *,"!"!£, EX VIVO tho therapy after adding patient's ceSs) 75 Department of Experimental Biology https://www.thegenehome.com/how-does-gene-therapy-work/techniq MUNI SCI Road Map of Gene therapy • In vivo delivery • In vivo gene therapy refers to direct delivery of genetic material either intravenously (through an intravenous) or locally to a specific organ (eg, directly into the eye). • In vivo gene therapy works through the help of a vector, which directly inserts functional copies of a gene into target cells to treat a mutated or missing gene. • In vivo delivery of gene therapy has been proven in many areas of research. Some of the currently approved gene therapies deliver genetic material in vivo. Targeted in vivo gene therapy will continue to evolve as scientists continue to refine methods of gene delivery. IN VIVO (the gene therapy is the therapy) functioning genetic material is packaged with a vector for delivery to patient's cells /- therapy is delivered to a patient's body either systemic-lly or locally to the specific organ COMMON ORGAN TARGETS: eye, brain, liver, skeletal muscle 76 Department of Experimental Biology https://www.thegenehome.com/how-does-gene-therapy-work/techniques MUNI SCI Road Map of Gene therapy EX VIVO (the gariD Ihorapy beeomni the therapy after adding patient's cell:) functioning gmolic material k jddcd !(i 1hc patients