; LOSCHMIDT , LABORATORIES Advanced molecular biology tools Molecular cloning and gene assembly in biotechnology, biomedicine and basic research Martin Marek Loschmidt Laboratories Faculty of Science, Masaryk University Kamenice 5, bid. C13, room 332 martin.marek@recetox.muni.cz Molecular Biotechnology (2023) 1 What is molecular biology? a The molecular biology studies biological macromolecules and the molecular mechanisms found in living systems, such as the molecular nature of the gene and its mechanisms of gene replication, mutation and expression. Front view Template DNA NCP m > LOSCHMIDT ' LABORATORIES Central dogma of molecular biology CENTRAL DOGMA : DNA TO RNA TO PROTEIN BYJU'S Pic inA| App DNA RNA Transcription Reverse transcription Replication Protein Translation © Byjus.com The central dogma of molecular biology states that DNA contains instructions for making a protein, which are copied by RNA. RNA then uses the instructions to make a protein. In short: DNA -> RNA Protein, or DNA to RNA to Protein. LOSCHMIDT LABORATORIES How DNA directs protein synthesis TRANSCRIPTION Codon Ribosome Redundancy of the genetic code • Degeneracy of codons is the redundancy of the genetic code, exhibited as the multiplicity of three-base pair codon combinations that specify an amino acid • The genetic code is degenerate mainly at the third codon position • The genetic code consists of 64 triplet codons specifying 20 canonical amino acids and 3 stop signals LOSCHMIDT LABORATORIES , LOSCHMIDT ; LABORATORIES A GUIDE TO THE TWENTY COMMON AMINO ACIDS AMINO ACIDS ARE THE BUILDING BLOCKS OF PROTEINS IN LIVING ORGANISMS. THERE ARE OVER 500 AMINO ACIDS FOUND IN NATURE - HOWEVER, THE HUMAN GENETIC CODE ONLY DIRECTLY ENCODES 20. 'ESSENTIAL'AMINO ACIDS MUST BE OBTAINED FROM THE DIET, WHILST NON-ESSENTIAL AMINO ACIDS CAN BE SYNTHESISED IN THE BODY. Chart Hey: ALIPHATIC AROMATIC ACIDIC BASIC HYDROXYLIC SULFUR-CONTAINING AMI DIC O NON-ESSENTIAL I I ESSENTIAL NAME O three tetter cade UNA cottons alanneQ Ala tit I, lit!, litA, ütli GLYCINE G Gly Mil. üüt.ütiA, ütiS isoleugineO lie Al I.Alt, A1A NH \ \ OH I LEUCINE O Leu t II. tit, tlA. t 10 I IA I It. PROLINE O Pro CCl. ttt, Ltfi, ttü I OH I NH, \ / _ *<* VALINE O Va! ÜI l.tjlt. O'VUt PHENYLALANINE Phe TTT.TTf TRYPTOPHAN Q Trp Ter, TYROSINE 7yr ASPART1CACIDQ GLUTAMIC ACID O TAT, TAC c;atp gat r~*A,r.AT, ARGININE O Arg ca, rcr, rr.A, rer.. .'«"„',. A;",r. / 1 € ii ^ HN-\ \ ° \ OH I HISTIDINE O \ o \ OH LYSINE O AAA, AAC SERINE 0 TfT, T This chart only shows those amino acids for which the human genetic code directly codes for. Selenocysteine is often referred to as the 21st amino acid, but is encoded in a special manner. In some cases, distinguishing between asparagine/aspartic acid and glutamine/glutamic acid is difficult, In these cases, the codes asx (B) and glx (Z) are respectively used. ©COMPOUNDINTEREST2014-WWW.COMPOUNDCHEM.COM | Twitter: @compoundchem | Facebook: www.facebook.com/compoundchem Shared under a Creative Commons Attribution-Noncommercial NoDcrivativcs licence. BY HC HQ LOSCHMIDT LABORATORIES What is genetic engineering? Genetic engineering is the action to modify the genetic information present in a living cell Adding, substituting or removing a genetic information in a given biological system necessarily implies to physically introduce a new information into the target cell. DNA is the physical support of genetic information. Therefore, genetic engineering relies on the generation of artificial DNA molecules, containing the information of interest, so it can be transferred to the target cells In molecular biotechnologies, genetic engineering represents a process of taking a gene from one species and putting it into another species Bacterium Human cell 1. Plasmid DNA O 2. DNA is cut with restriction enzymes. 3. Plasmid is reintroduced into bacterium. 4. Engineered bacteria multiply producing insulin. '* # , 5, Insulin is separated and purified to produce human insulin. DNA Human insulin-producing gone Bacterial DNA with human gone inserted -Human insulin Insulin injected into patient Applications of molecular biotechnologies LOSCHMIDT LABORATORIES Molecular biology in rational (computational) protein design Computational protein design i • De novo DNA synthesis • Gene assembly • Site-directed mutagenesis Gene synthesis & cloning 5. • Biocatalysis • Binding assays • Protein-protein interaction • Photoactivation Biological assays NMR spectroscopy Cryo-EM X-ray crystallography • Expression host • Affinity and solubility tags • Buffer screening Protein expression & purification 3. Biophysical & biochemical characterization 4. 3D structure determination • CD spectroscopy • Differential scanning fluorimetry • Dynamic light scattering •SAXS LOSCHMIDT LABORATORIES The key role of protein production in pharma industry 1U <íu i i ISVG I VYGDQYRQLCCSSPKFGDF IS I G I VYGDQYRRVCCSSPKFGDF ISVG IVYGDQYRRLCCSSPKFGDF PVGIVYSSEIHRLSDLSPKYGNF Identify target (Genomics, biological assays) Isolate protein involved in disease (Molecular cloning, recombinant expression, biochemical and structural analyses) 038 Find a drug effective against disease protein (virtual screening, HTS and library screening etc.) 1 dft± 0,5 |j[ Preclinical testing (Animal studies, scale-up, formulation) Human clinical trials Protein production is a significant bottleneck in early phase drug discovery FDA approval Bdeodoq* IMinoslatl lor mjKiiw 500 mg/vial Smjta-UsiVM Piícjľfl Unuwd Ponw^ Recombinant protein production workflow • Gene synthesis and molecular cloning • Protein expression • Protein purification • Protein characterization • (Protein structure determination) LOSCHMIDT LABORATORIES DNA synthesis & molecular cloning Concepts Methods Applications 12 Structure of DNA B Thymine Nitrogenous \ Base 0 11 HO-P-O-n 1 O" Phosphate N OH Deoxyribose Sugar H2N Adenine Adenine Phosphodiester A) A single nucleotide. The phosphate and deoxyribose sugar form the backbone of DNA. The nitrogenous base (in this case adenine) is the information-carrying unit of each nucleic acid. B) The structure of single-stranded DNA. In nature, enzymes form phosphodiester bonds (blue circles) that link the 5th position and 3rd position of adjacent deoxyribose sugars. Due to the modular nature of nucleotides, this chain can grow indefinitely. OH Deoxyribose sugar Chemical synthesis of DNA ■ Making DNA chemically rather than biologically was one of the first new technologies to be applied by the biotechnology industry. The ability to make short synthetic stretches of DNA is crucial to using DNA replication in laboratory techniques. DNA polymerase cannot synthesize DNA without a free 3'-OH end to elongate. Therefore, to use DNA polymerase in vitro, the researcher must supply a short primer. Such primers are used to sequence DNA, to amplify DNA with PCR, to introduce DNA mutations, and even to find genes in library screening. ■ Technically, oligonucleotides are any piece of DNA approx. 20 nucleotides in length, but today oligonucleotide denotes a short piece of DNA (approx. 125 nucleotides) that is chemically synthesized. ■ Unlike in vivo DNA synthesis, artificial (chemical) synthesis is done in the 3' to 5' direction. \ j> ■ iPr dA-CE-phosphoramidite 5'-dimethoxytrityl-H-benzoyl-2-deo)(yadenosine-3'-[(2-cyanoethyl)-(N:N-diisopropy)]-phosphoramidite LOSCHMIDT LABORATORIES Overview of the phosphoramidite approach HO Step 1: deprotection Acid-catalyzed removal of DMT allows for subsequent base addition. C,G) DMTOv o R (ATCG) Step 2: base coupling A DMT-protected phosphoramidite is added to the unprotected 5' OH using a tetrazole activator. ov o p N(iPr), Step 3: capping (optional) Unreacted 5' OH are acetylated to prevent further chain extension. This step helps prevent single-base deletions at the expense of yield. 1 (A,T,C,G) Step 4: oxidation Oxidation of phosphite triester to phosphate using aqueous iodine This 4-step cycle repeats until the oligo receives its final nucleotide. LOSCHMIDT LABORATORIES Automated and miniaturized oligonucleotide synthesis Base A .- G ^ j^^^ T ^ Capping Start (synthesis platform) Coupling ■T coupling cycle can be repeated to extend the oligonucleotide molecule in a desired sequence. The schematic view of the oligonucleotide synthesis Microreactor chip Single microreactor Oligonucleotide synthesis (a) The synthesis processes involve designing target sequence, delivering chemical reagents through the inkjet printer, and oligonucleotide synthesis in the microreactor chip, (b) The single microreactor is filled with silica beads which enhance the surface area for the following synthesis. The beads are inherently fixed in the microreactor using sintering process, (c) The oligonucleotide synthesis on the silica beads follows the four-step with phosphoramidite strategy: deprotection, coupling, capping and oxidation. LOSCHMIDT LABORATORIES Enzymatic DNA synthesis: polymerase chain reaction (PCR) DNA primers DNA polymerase Step 1: denaturing (95 °C) parent DNA DNA template strand -\- repeat cycle (20-40 times) - 3'C two DNA strands new DNA strands four DNA strands Taq nucleotides (dTTP, dCTP, dATP, dGTP) Step 2: annealing {55 X) 5 | 5' 3 I p I 5-I section of DNA to be amplified 3' P 5' Taq 3' 3' 5' 5' I P III Taq TJ W 3 (I> « n" 3' CD o O © Encyclopedia Britannica, Inc. Amplification of up to 20 kbp DNA fragment from pre-existing template (genomic loci, cDNA library, cloned fragment etc.) Gel electrophoresis Power Supply (80-120V) o DNA Sample loading XT DNA Sample in gel loading buffer DIMA Separation Application of Electrical Field (DC) Anode t = 30-45min Marker DNA Samples DNA 1 I fr-"- Separated DNA Fragments UV transillumination & ^Documentation Gel electrophoresis is a method for separation and analysis of macromolecules (DNA) and their fragments, based on their size and charge. It is used in molecular biology to separate a mixed population of DNA fragments by length, to estimate the size of DNA fragments. Different PCR protocols use 1. AFLP PCR 2. Allele-specific PCR 3. Alu PCR 4. Assembly PCR 5. Asymmetric PCR 6. COLD PCR 7. Colony PCR 8. Conventional PCR 9. Digital PCR (dPCR) 10. Fast-cycling PCR 11. High-ftdeNty PCR 12. Hot-start PCR 13. In situ PCR 14. Intersequence-specific (ISSR) PCR 15. Inverse PCR 16. LATE (linear after the exponential) PCR 17. Ligation-mediated PCR 18. Long-range PCR IIIIIIMII l 19 Methylation-specific PCR (MSP) 20. Miniprimer PCR 21. Multiplex-PCR 22. Nanoparticle-Assisted PCR (nanoPCR) 23. Nested PCR 24. Overlap extension PCR 25. Real-Time PCR (quantitative PCR or qPCR) 26. Repetitive sequence-based PCR • 27. Reverse-Transcriptase (RT-PCR) 28. Reverse-Transcriptase Real-Time PCR (RT-qPCR) 29. RNase H-dependent PCR (rhPCR) 30. Single cell PCR 31 Single Specific Primer-PCR (SSP-PCR) 32. Solid phase PCR 33. Suicide PCR 34. Thermal asymmetric interlaced PCR (TAIL-PCR) 35. Touch down (TD) PCR 36. Variable Number of Tandem Repeats (VNTR) PCR Potymors» ch-am rwcEion - PCR 1 _ 1 ...iiiiii; ' • liumm , ,„.l.i!m ^* Real-time PCR / quantitative PCR (qPCR) ■ It is a technique used to monitor the progress of a PCR reaction in real-time. ■ At the same time, a relatively small amount of PCR product (DNA, cDNA or RNA) can be quantified. ■ The process is monitored in "real-time". The reaction is placed into a real-time PCR machine that watches the reaction occur with a camera or detector. ■ To link the amplification of DNA to the generation of fluorescence which can simply be detected with a camera during each PCR cycle. ■ Hence, as the number of gene copies increases during the reaction, so does the fluorescence, indicating the progress of the reaction. LOSCHMIDT LABORATORIES Digital PCR (dPCR) Sample dilution and PCR reaction mix setup Blue - Target Red - Background (gDNA, cDNA; primers/probes; master mix) boooooooq JOOOOOOOO ooooooooc JOOOOOOOO ooooooooc JOOOOOOOO ooooooooc J o o o o o o o o ooooooooc ?oooooooo PCR reaction partitioning into thousands of individual reactions OOUOOO&. oo©ooooc )OOOO0®00 OOOOOOOOC :ooOOOOOO ooooooooc 500®00®00 ooooooooc 50000000G ooooooooc ;oooooooo igogoo^o\P>p>y End-point PCR amplification of partitions Readout and absolute quantification ■ Digital PCR is a highly precise approach to sensitive and reproducible nucleic acid detection and quantification. ■ Measurements are performed by dividing the sample into partitions, such that there are either zero or one or more target molecules present in any individual reaction. ■ Each partition is analyzed after end-point PCR cycling for the presence (positive reaction) or absence (negative reaction) of a fluorescent signal, and the absolute number of molecules present in the sample is calculated. It does not rely on a standard curve for sample target quantification. ■ Eliminating the reliance on standard curves reduces error and improves precision. LOSCHMIDT LABORATORIES Comparison between real-time qPCR and digital PCR Real-time qPCR Bulk reaction analysis Relative quantification; C Digital PCR ^ « ^ « 4J «y V " O *0> Random distribution of molecules into partitions Absolute quantification: Copies/|jl LOSCHMIDT LABORATORIES Reverse transcription polymerase chain reaction (RT-PCR) Incubate with reverse transcriptase to synthesize cDNA strand A-A-A-A31 mRNA 3' T-T-T-T 5' Oligo(dt)primer 11II111111IIIIIIII mi mRNA cDNA When cDNA strand is completed hydro I/ze RNA stand IHIMIIII III! Iirilll II CDNA Conversion of RNA into cDNA using reverse transcriptase Amplification of cDNA using PCR cDNAis DNAthat is synthesized from messenger RNA molecules. cDNA synthesis is catalyzed by an enzyme called reverse transcriptase, which uses RNA as a template for DNA synthesis. Reverse transcriptase was initially discovered and isolated from a retrovirus. These viruses contain an RNA genome; therefore the viruses need to produce a cDNAcopy of their genome to be compatible with the host cell's molecular machinery. Plasmids: essential tools for genetic engineering LOSCHMIDT LABORATORIES What is a plasmid? how do I know that the cells I'm growing actually contain my vector? SELECTION MARKER antibacterial resistance gene if it grows, you knows! PROMOTER start making RNA here OF WHAT? ORIGIN OF REPLICATION says start copying the DNA here mylNSERTed cDNA! how'd that get in here? <5° I cut & pasted at these RESTRICTION SITES what if I need more plasmid? PLASMIDS are "extrachromosomal" (not part of the chromosomes), circular pieces of DNA. Similarly to chromosomes, they are double-stranded, which means they can easily be "unzipped" and copied (replicated). Plasmids use the host's machinery (DNA polymerase), but they don't have to wait for the host to divide to copy themselves —► lots of copies of themselves. When the cell does divide, these copies will get split between the daughter cells, so they'll inherit the plasmid as well. The plasmid can act as a VECTOR - a vehicle for taking genes we want to deliver into cells. Always sequence your plasmid to double-check that the gene is correctly inserted LOSCHMIDT LABORATORIES Antibiotics as selectable markers try to put your plasmid into ^bacteria host cell DNA doesn't have the resistance gene plasmidsdo +o l and other Beta-lactams target cell wall synthesis use antibiotics to select for those that actually took it TETRACYCLINE KANAMYCIN , and other tetracyclines ana other aminoglycosides oh o HO h o o & H3N5——-i—- NH2 both target protein synthesis (translation) but in different ways Restriction endonucleases ■ Restriction enzyme, also called restriction endonuclease, is a protein produced by bacteria that cleaves DNA at specific sites along the molecule. ■ Restriction endonucleases cut the DNA double helix in very precise ways. It cleaves DNA into fragments at or near specific recognition sites within the molecule known as restriction sites. They have the capacity to recognize specific base sequences on DNA and then to cut each strand at a given place. Hence, they are also called as 'molecular scissors'. LOSCHMIDT LABORATORIES Restriction endonucleases (restriction enzymes) I lindlll ^ s- • digest 5' protruding ends Pstl ^ 5' digest 3' ami- 5' ^ 3 35 3' [ 3 5 3' protruding ends digest a^B5' s'l J6' Blunt ends Type I enzymes cleave at sites remote from a recognition site; require both ATP and S-adenosyl-L-methionine to function; multifunctional protein with both restriction and methylase activities. Type II enzymes cleave within or at short specific distances from a recognition site; most require magnesium; single function (restriction) enzymes independent of methylase. Type III enzymes cleave at sites a short distance from a recognition site; require ATP (but do not hydrolyze it); S-adenosyl-L-methionine stimulates the reaction but is not required; it exists as part of a complex with a modification methylase. Type IV enzymes target modified DNA, e.g. methylated, hydroxymethylated and glucosyl-hydroxymethylated DNA. Restriction cloning The production of exact copies of a particular gene or DNA sequence using genetic engineering techniques is called gene cloning. LOSCHMIDT LABORATORIES PCR-based cloning EcoRlV Ari'fJ XhDl -VEcoRI -Hindlft Natl Digest Nütl Digest EwRI N.::l KcLi ^ EcoRl Insert (VGOI) .— Notl Ligate Xhol EcoRI Insert (VGOI) Not I Am:; Amp Amp ■ DNA fragment (gene) of interest can be flanked by any restriction site 3, LOSCHMIDT LABORATORIES Golden gate DNA assembly Bsal-HFv2 5 GGTCTCNiN N N N a1 a CCAGAGNNNNNi 5 P1 4^ 1 P2 \ 1 GGTCTCNJUUVI jkUUUIJMGAGACC CCAGAGNMitf NN NN!N CTCTG6 P4 PCR amplification of fragments B Single-tube reaction • Bsal-HFv2 • DNA ligase PCR-amplified fragments, Bsal-HFv2-digested Can be used in complex (20+) fragment assemblies Simultaneous and directional assembly of multiple DNA fragments into a single piece using Type lis restriction enzymes and T4 DNA ligase. LOSCHMIDT LABORATORIES Sequence and ligation independent cloning (SLIC) Plasmid PCR T4 DNA Polymerase {3' exo) Anneal j SLIC, or sequence and ligase independent cloning, does not utilize restriction enzymes or ligase. A DNA sequence fragment to be cloned into a destination vector is PCR amplified with oligos whose 5' termini contain about 25 bp of sequence homology to the ends of the destination vector, linearized either by restriction digest or PCR amplification. Transform LOSCHMIDT LABORATORIES Gibson DNA assembly + DIMA inserts wilh 15-20 bp overlapping ends (PCR-amplilied) Incubate at 50 C lor 15-SQ minutes Why Gibson Assembly? No need for specific restriction sites. Join almost any 2 fragments regardless of sequence No scar between joined fragments. Fewer steps. One tube reaction. Can combine many DNA fragments at once. Single-tube reaction * Gibson Assembly Master Mix - 5' eKonuclease - DNA polymerase - DNAhgase NEB Gibson Assembly Cloning Kit (NEBtfE551fl) ' Gibson Assembly Master Mix (NEB 0E2611) ■ NEB5-alpha Cornpetenl E. coti (NEB Transformation and plating LOSCHMIDT LABORATORIES Traditional restriction cloning versus Gibson assembly Traditional Restriction Cloning i Digest Fragment 4 Linearize Plasmid i 4 Dephosphorylate Ends Rungeland 4 Isolate fragment Purify Plasmid 4 4 Quantify molar amounts of fragments Combine and perform overnight ligation Transform competent cells and plate on selective media Repeat step to include additional inserts k Pick colonies and screen Gibson Assembly3 Method Linearize Plasmid I Purify Plasmid I Generate Gibson fragments by PCR, gene synthesiser restriction digestion 1 Combine and perform 60-80 minute Gibson Assembly Procedure Single reaction to include one or multiple inserts 4 Transform competent cells and plate on selective media 4 Pick colonies and screen Large-scale de novo DNA synthesis ■ Protein engineering ■ Engineered metabolic pathways ■ Synthetic biology ■ Whole-genome syntheses ■ DNA nanotechnology (DNA computers) LOSCHMIDT LABORATORIES DNA synthesis prices 100 10 0.1 Price Per Base of Synthetic DNA Rob Carlson, February 2D 14, www.synthesis.ee 0.01 1990 Cosl ShdrtOhgo Cost: Gene Synthesis 1995 2TM 2CC5 2C1C 2015 Year LOSCHMIDT LABORATORIES DNA mutagenesis Concepts Methods Applications 38 LOSCHMIDT LABORATORIES Site-directed mutagenesis Site-directed mutagenesis is used to generate mutations that may produce a rationally designed protein that has improved or special properties (i.e. protein engineering). The basic procedure requires the synthesis of a short DNA primer. This synthetic primer contains the desired mutation and is complementary to the template DNA around the mutation site so it can hybridize with the DNA in the gene of interest. The mutation may be a single base change (a point mutation), multiple base changes, deletion, or insertion. The single-strand primer is then extended using a DNA polymerase, which copies the rest of the gene. The copied gene thus contains the mutated site, and is then introduced into a host cell in a vector and cloned. Finally, mutants are selected by DNA sequencing to check that they contain the desired mutation. 39 3, LOSCHMIDT LABORATORIES Site-saturation mutagenesis (SSM) Site saturation mutagenesis is used to substitute targeted residues to any other naturally occurring amino acid The core of a SSM experiment lies in the codon degeneracy or randomness. A completely randomized codon (NNN, where N=A, C, G or T) results in a library size of 64 different sequences encoding all 20 amino acids and 3 stop codons When an experiment targets multiple codons, the library size can be considerably higher, making it difficult to perform a complete screening (e.g. targeting three NNN codons has 262,144 unique codon configurations Nonpolar aliphatic Potai uncharged Bulky aromatic Polar charged 1814 Ü815 D816 Q817 A818 V819 R820 T821 A822 S824 G825 Y826 M827 0828 R829 R830 G A V L MIST C P N Q F Y w K R H D E mm X m ■ ■ ITS X X mm X ■i X x i mm 1- ■ - X ■ -- 'SB UM X x mm x l 20 40 W 30 10D 120 140 160 teo 200% Loss of Function Gain of Function 40 LOSCHMIDT LABORATORIES Site-saturation mutagenesis (SSM) Starting gene Library Individual library members 1) PCR gene with degenerate primers 2) Assemble into plasmid 41 LOSCHMIDT LABORATORIES Error-prone PCR (epPCR) PCR Error-Prone PCR ............T~ IIJILIIIII ~ dCTP, dTTP dCTP,dTTP t dGTP, dATP dGTP, dATP 4, Mg2+ Mg2+ t Mn2+ The error rate of Taq DNA polymerase is 0.001-0.002 % per nucleotide per replication cycle under standard conditions which is sufficient to create mutant libraries of large genes but not for small genes Error-prone PCR (epPCR) takes advantage of the inherently low fidelity of Taq DNA Polymerase, which may be further decreased by the addition of Mn2+, increasing the Mg2+ concentration, and using unequal dNTP concentrations. The rate of mutagenesis achieved by error-prone PCR is in the range of 0.6-2.0 % https://lifescience.canvaxbiotech.com/product/pickmutant-error-prone-pcr-kit/ 42 LOSCHMIDT LABORATORIES Mutator strains Mutator strains of E. coliare deficient in one or more of DNA repair genes, leading to single base substitutions at a rate of approximately 1 mutation per 1000 base pairs Generation of mutant libraries Process is simple DNA polymerase Original (template) DNA Replication fork i > Adenine t < Thymine (=> Cytosine Guanine I I I II I DNA polymerase Original (template) DNA strand 43 LOSCHMIDT LABORATORIES Insertion and deletion (InDel) mutagenesis Gain or lost of one or more nucleotides produces frameshift mutations (triplet reading frame) Triplet InDel mutagenesis may trigger protein backbone changes essential for evolvability Insertion and deletion mutations can enhance proteins through structural rearrangements not possible by substitution mutations alone A Trinucleotide Deletion Directed evolution Obp( V V zn w w w I ▲ AiltAJtlA AÄJL^i A kä äk AJ 1 I 720 bp Beneficial mutations Using directed evolution, green fluorescent protein (GFP) was observed to tolerate residue deletions, particularly within short and long loops, helical elements, and at the termini of strands. A variant with G4 removed from a helix (EGFPG4A) conferred significantly higher cellular fluorescence. Arpino et al., Structure 22: 889-898 (2014) 44 LOSCHMIDT LABORATORIES DNA shuffling DNA shuffling is a method for in vitro recombination of homologous genes The genes to be recombined are randomly fragmented by DNasel, and fragments of the desired size are purified from an agarose gel These fragments are then reassembled using cycles of denaturation, annealing, and extension by a polymerase Recombination occurs when fragments from different parental templates anneal at a region of high sequence identity Following this reassembly reaction, PCR amplification with primers is used to generate full-length chimeras suitable for cloning into an expression vector Moving from DNA shuffling to whole genome shuffling is known as GENOME SHUFFLING Homolog 1 Homolog 2 Homolog 3 Homologous genes Fragmented genes Chimeric genes / \ t Screen for interesting variants 45 LOSCHMIDT LABORATORIES The CRISPR/Cas9 system • The Cas9 (CRISPR associated protein 9) is a protein which plays a vital role in the immunological defense of bacteria against DNA viruses, and which is used in genetic engineering. Its main function is to cut DNA and therefore it can alter a cell's genome • Structurally, Cas9 is an RNA-guided DNA endonuclease enzyme associated with CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) adaptive immunity system in Streptococcus pyogenes • Cas9 performs this by unwinding foreign DNA and checking for sites complementary to the 20 bp spacer region of the guide RNA • If the DNA substrate is complementary to the guide RNA, Cas9 cleaves the invading DNA Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA dsDNA Targel Cleava^e -A VI Crystal structure of S. pyogenes Cas9 in complex with sgRNA and its target DNA at 2.5 A resolution, Nishimazu etal., Cell 156: 935-49 (2014) 3, LOSCHMIDT LABORATORIES The CRISPR/Cas9 system: key elements Cas9 nuclease specifically cleaves double-stranded DNA activating double-strand break repair machinery In the absence of a homologous repair template non-homologous end joining can result in indels disrupting the target sequence Alternatively, precise mutations and knock-ins can be made by providing a homologous repair template and exploiting the homology directed repair pathway expression vector 1 ■ ■ single guid e (sg) RNA mammalian «RNA CrSCfRNA _ GNNNNNNNNNNNNNNNNNqG T11 t p 11 r I I r 11 I 11 M 11 jCNNNNNNNNNNNNNNNNNcC s or Insertion/ f;p Hli;;il —i New DNA dsDNA Target Cleauase >AM Coro- DNA Mew DNA Hon-homo logo us end joining (NHEJ) H ü I no I ü g v direcled repair (HDH) LOSCHMIDT LABORATORIES Advantages of CRISPR/Cas9-mediated mutagenesis The CRISPR/Cas9 system requires only the redesign of the crRNAto change target specificity This contrasts with other genome editing tools, including zinc finger and TALENs, where redesign of the protein-DNA interface is required Furthermore, CRISPR/Cas9 enables rapid genome-wide interrogation of gene function by generating large gRNA libraries for genomic screening ......1 M ZFNs D ■■ ■■ ■■ ■■■■■■ ■■ ■■ ■■ ■■ ■■ ■■ ■■ ■ T^TTTT? i ■ ■■ ■■■■■■■■■■■■ ■ ■ ■ ■ Zinc-finger nucleases ALEs n .......... FCn.i JJ.i.U.U.UJJJJ.i.U.U q:iie TALENs r 3.V1.1 MM 3 JJJTlVVVVM J 3 J11.1.1.1 VIJ 3 I I I I rJ L / sgRWA (racrRNA Cas9 The mini-RNA-guided endonuclease CRISPR-Cas12j3 • CRISPR-Cas12j is a recently identified family of miniaturized RNA-guided endonucleases from phages. • These ribonucleoproteins provide a compact scaffold gathering all key activities of a genome editing tool. • A site-directed mutagenesis analysis supports the DNA cutting mechanism, providing new avenues to redesign CRISPR-Cas12j nucleases for genome editing. nature communications Explore content v About the journal w Publish with us v nature > nature communications > articles > article Article | Open Access | Published: 22 July 2021 LOSCHMIDT LABORATORIES Molecular carpentry DNA polymerases DNases Exonucleases Restriction endonucleases Kinases Phosphatases Ligases Etc. cut insert (what you want to put in) and vector (home you want to put it in) with the same 2 restriction enzymes EcoRI * IT the rest BamHI EcoRI QP 5'. . G GATCC... 3' 5'.. . GJAATTC. 3'. . CCTAG G... 5' 3'.. CTTAAJG. EcoRI BamHI This generates DNA pieces with complementary/'sticky ends"you can mix & match (once you separate them) .G3' 5'GATCC... . CCTAG 5' 3'G... ...G3' 5'AATTC. ...CTTAA5' 3'G. .G3' 5'GATCC. .CCTAG 5' 3'G. ,G3' 5'AATTC. . CTTAA 5' 3'G. purify the pieces you want run an agarose gel to separate by size, then cut out & purify those you want mix em v > + 5> stitch 'em DNA ligase I wish I could report otherwise, but the cloning is not going very well. 51 LOSCHMIDT LABORATORIES Molecular biology in protein technologies Computational protein design i • De novo DNA synthesis • Gene assembly • Site-directed mutagenesis Gene synthesis & cloning 5. • Biocatalysis • Binding assays • Protein-protein interaction • Photoactivation Biological assays • Expression host • Affinity and solubility tags • Buffer screening Protein expression & purification 3. Biophysical & biochemical characterization 4. NMR spectroscopy Cryo-EM X-ray crystallography 3D structure determination • CD spectroscopy • Differential scanning fluorimetry • Dynamic light scattering •SAXS Molecular Biotechnology Practicals Kdy: Středa 11.10. 2023 Od 9:00 (skupina I.) Od 13:00 (skupina II.) Kde: C13-332 53 LOSCHMIDT LABORATORIES Questions 54 LOSCHMIDT LABORATORIES Supplementary materials 55 LOSCHMIDT LABORATORIES Gateway cloning The GATEWAY Cloning Technology is based on the site-specific recombination system used by phage A to integrate its DNA in the E. coli chromosome. Both organisms have specific recombination sites called atP in phage A site and atB in E. coli. The integration process (lysogeny) is catalyzed by 2 enzymes: the phage A encoded protein Int (Integrase) and the E. coli protein IHF (Integration Host Factor). Upon integration, the recombination between atB (25 nt) and attP (243 nt) sites generate aflL (100 nt) and aftR (168 nt) sites that flank the integrated phage I DNA. The process is reversible and the excision is again catalyzed Int and IHF in combination with the phage A protein Xis. The atlL and aftR sites surrounding the inserted phage DNA recombine site-specifically during the excision event to reform the atP site in phage A and the atB site in the E. coli chromosome. Integration attP ZZ arts (POPJ) (BOB') A Phage Chromosome. am n (BOP') attR (POBr) Chromosome Chromosome I ntegrati on orti I 1 Excision Integrated prophage LOSCHMIDT LABORATORIES Gateway cloning The GATEWAY reactions are in vitro versions of the integration and excision reactions. To make the reactions directional two slightly different and specific site were developed, att\ and att2 for each recombination site. These sites react very specifically with each other. For instance in the BP Reaction affB1 only reacts with a#P1 resulting in affl_1 and affFM, and a#B2 only with a#P2 giving affl_2 and a#R2. The reverse reaction (LR Reaction) shows the same specificity._ 3ÍÍP1 3HB1 Gene of interest attB2 attP2 ař/L1 BP reaction affL2 1 afiL1 afíL2 aftR1 atfR2 pDestination vector LR reaction LOSCHMIDT LABORATORIES Gateway cloning ařf P1 ařřB1 Gene of interest aířB2 aříP2 aííL1 |[ pDonor vector BP reaction afřL2 1 ař/L1 aířL2 ařřR1 afíR2 LR reaction ■ Lambda bacteriophage site specific integration system LOSCHMIDT LABORATORIES SLIC Sequence and Ligation Independent Cloning start with PCR to make copies of insert piece & vector piece with overlapping regions on the ends something with insert (such vector you want to put it in, as gene) you want such as a plasmid design your PCR primers to have bits of the flanking vector sequences on their ends note: anything can be in the purple part or gray part - we're not copying those parts so they don't matter add Dpnl to degrade leftover parent plasmids ^ it only cuts methylated DNA - parent plasmids are methylated but PCR products aren't ti. \\ use T4 polymerase to chew the ends back (a little) so they'll stick1 this exposes complementary overhangs mix pieces together >-*-*""\& stick in bacteria to do the rest homologous recombination machinery will fill in the gaps no need to pre-ligate LOSCHMIDT LABORATORIES PCR copying DNA the (tiny) test tube way! \ », ■ ■ ■ y i \-^. ~f ■>. w m v. v v v< j . ^ | ° ^chYaSe design primers (1 per strand) - short DNA sequences Reaction bookending the region of DNA you want copied 5' melt - raise temp to separate strands each 5- cycle 3- 3' 5'. then anneal - lower temp so primers can bind 5' 3'1 ,3' 3' and extend - let the DNA Pol add matching bases 1st cycle end where Pol runs out of steam or time or falls off 5', 3' 3'1 5' 3' 5' now when you melt, these newly-made strands can get used as templates 2nd cycle 3' 5' end where 1st primer started because 3' "3 template stops there & Pol falls off do it again and again and again all future cycles amplify pieces bookended by the primer start sites 51 5'. 3' 5'. 3' 3' 5' 5'. 3'" 5'. 5. 3' 5 3 .3' 5 5' 3 3' 5' .3' 5' 3' 5' .3' 5' .3' 5' _3' '5' .3' 5' Cloning Controls antibiotic resistance gene in the vector aliows for antibiotic selection -grow with that antibiotic so only bacteria with the vector can grow uncut vector positive control that transformation worked & cells are competent (able to take in DNA) each colony's made up of genetically-identical bacteria (hopefully with your insert) cut vector negative control that all vector got cut so you didn't have any "parent" vectors & or self-circularized vector also shows that the antibiotics were effective Gateway cloning The GATEWAY Cloning Technology is based on the site-specific recombination system used by phage A to integrate its DNA in the E. coli chromosome. Both organisms have specific recombination sites called atP in phage A site and atB in E. coli. The integration process (lysogeny) is catalyzed by 2 enzymes: the phage A encoded protein Int (Integrase) and the E. coli protein IHF (Integration Host Factor). Upon integration, the recombination between atB (25 nt) and attP (243 nt) sites generate aflL (100 nt) and attR (168 nt) sites that flank the integrated phage I DNA. The process is reversible and the excision is again catalyzed Int and IHF in combination with the phage A protein Xis. The attL and attR sites surrounding the inserted phage DNA recombine site-specifically during the excision event to reform the atP site in phage A and the atB site in the E. coli chromosome. DNA Polymerase does this growing chain longer chain e BASE On A5E OH free 3'OH ©§©, H 5 \0 © ©0 © BASE + ©O© OOOOOO freeS' ©- ©- o o © BASE "© I©©© 0- 0- triphosphate V BASE OH © ©1 5- loj pyrophosphate (PP,) DNA Lig broken (or gapped) chain © BASE ATP © BASE T""^ does this continuous chain OH free 3'OH © BASE © O free 5' © \£j monophosphate T AMP H%. - £ On/' ©O©- N © \£j OH OH o © © © © 0- 0- pyrophosphate (PP5) LOSCHMIDT LABORATORIES The CRISPR/Cas9 system on YouTube https://www.youtubexom/watch?v=bXnWlk8FgKc https://www.youtubexom/watch?v=OjNrbPMXyMA https://www.youtube.com/watch?v=0dRT7slyGhs https://www.youtube.com/watch?v=2pp17E4E-08 totr/ttlrtl IMint J-Wr HVUrt^ Mif^ld. - ■.'—.>TihiwinWntfi-t',,'YrlTJl' -^fl O B S3 * 1 t 4 (J* □ = ^ FN|wrnmiJti ohltdi/oiH^ Google = DfauTita" crrprscaBir 2 DM /il rtiWnti'i - l'ubl*«wiTOi M 2011 rAlPOMFHflUT pozwji zkowtrolovat Q pfilHLASITH What you [MrfioJcnoiniaDOJi ^RISPflifllHlJdfgHlMIl TIM MUKl' J' (iaiiDOf L-IP 311 Ira iMechuli i: 1= - = : ■- = !:- ■- .--5 ■■■■==■: Earth - HieBBCterloplHge KLTTnHui - In n WiitsJvJ S Rlot«*nfjliviv MFnor«finfl nqy I AimIp™h«iH*il | KirartibiilyLf rlKrjma'H Cas'*3¥slenH3an 4ih> Lif- 3< ■fr 14 ns. f U fi&fiit itdirt ■ rfikttn.il. How to triple your mcmw^ try using thistTicJt | RlcarriDL Isiiw TFCViTaft:? A LOSCHMIDT LABORATORIES recom bina nt DN A/prptein molecular take a segment of DNA from its home & stick it cloning into a piece of DNA that's easier to work with since you've "recombined" DNA, we call this recombinant DNA when we do this with DNA containing protein instructions, the protein made from it is referred to as recombinant protein .......................................................................O GENE transcription mRNA translation^ PROTEIN RNA Pol post-transcription editing gDNA genomic DNA 5'cap mature mRNA poly(A) tail messenger RNA riboson _ _. _ DNA version of the edited copy (mRNA) we put CDNA complementary DNA into a vector plasmid for protein expression a common place to put the DNA is a circular piece of DNA called a plasmid vector plasmids have some key features... + a plasmid can act as a vector ^ ("vehicle") for getting that DNA into 1^ bacterial cells r^e note: we can use different vectors for different cell types/organisms selection marker promoter - allows for, transcription & subsequent translation /such as antibiotic resistance ' gene - allows for selection for IJJp \ cells with your plasmid DNA Pol ORI - allows for copying of plasmid since we control what DNA we put in, we can make changes to it to "custom-make" proteins, add tags for easier purification, etc. GENE transcription mRNA translation^ PROTEIN RNA Pol post-transcription editing "-5'cap poly(A)tail gDNA mature mRNA ribosomes genomic DNA messenger RNA _ DNA version of the edited copy (mRNA) we put CDNA complementary DNA jnt0 a vector p|asrnid for protein expression you can use use PCR to make lots of copies of the insert something with ^^^^m^ and you can use the primers to add on cut sites you want this cutting leaves you with phosphoryloted ends but the primers are usually synthesized without phosphates - this only comes into play if your vector is dephosphorylated LOSCHMIDT LABORATORIES dephosphorylation/phopshorylation may be needed if you're using a single REase, you'll need to dephosphorylate your vector to prevent self-circularization this now has sticky ^^*\^ ^/ ends for itself EcoRI 4& 5, G;AATTC 3, so when you add ligase, 3'...cttaa;g...5' it can self-liqate & since the antibiotic resistance gene is in there, it can survive - but it doesn't have your gene you can prevent self-circularization by dephosphorylating the ends but you need your insert to be phosphorylated! you7/ still have a couple of nicks, which the bacteria can fix + phosphatase v now ligase can't seal it Dr. Martin Marek Loschmidt Laboratories Faculty of Science, MUNI Kamenice 5, bid. A13, room 332 martin.marek@recetox.muni.cz L LOSCHMIDT ; LABORATORIES