Biotechnology of drugs - Basics of genetic engineering II. Cloning Classic definition - creating a new individual genetically identical to the original Biotechnology definition -fusion of vector with gene —► creation of genetically identical cells/organisms carrying vector with insert Human clone Gene clone Cell clone v| M M v| insulin. O transgenic bacterium with plasmid containing insulin gene Recombinant bacteria gain ability to secrete human insulin. Cloning vector Foreign DNA https://www.news-medical.net/life-sciences/The-Biochemistry-of-Cloning.aspx Ends of vector and foreign DNA fragment anneal and are mixed with DNA ligase Recombinant DNA Basic steps in gene cloning J 1) Cleavage of DNA at desired sites 2) Recombination - connection of DNA fragments 3) Transformation - insertion of recombined DNA into a cell 4) Selection of cells containing a foreign gene 5) Analysis of cloned DNA Transformed bacteria colonies turn white Gene of interest Untransformed bacteria Bacteria colonies transformed with empty vector turn blue S;/>#/B sub-cloned m pi I -2 I a( • and propagated in E. coli BI.-21 CodonPlus(DK3)-RIPL 3-D structure of JhBglB II Mil l-r"U00ü<<04u hV< it ReeonihiiuuH E. coli BI.2I CodonPlus C/3 0! ? s 7 -° LO O S2 w E £ 8 g> CO 03 I ? (D O) y c .5 o -o ö a> ■ = n zz P <-> = 75 £^ *= (U ^5 O "> £ o. 92 ts 'o SZ 03 Ligation = covalent joining of a vector to a fragment Nick 'TOoh 'p Up L-3t Nicked DNA Ligase-AMP -•- Ligase PPi ATP AMP S H "pvJ_J^PvJ_J^Pv|_pF AMP -P L^P Nicked DNA is joined 5 3' . 5' 3' OH • • • GA CTTA • • • GA ATTC // CTTA AG joining by ligase + 2 x ATP ATTC • • • AG . . . OH spontaneous connection 3' • • • *^ 3' 5' 5'... GAATTC ... 3' 3'... CTTAAG ... 5' Creation of overhanging ends - linkers ( 5'... CCGAATTCGG 3'... GGCTTAAGCC ligation 3' 5' target DNA target DNA 7A^2 CCGAATTCGG;target DNA GGCTTAAGCC: target DNA CCGAATTCGG CCGAATTCGG GGCTTAAGCC GGCTTAAGCC EcoRI T T 5' . . 3' . . AATTCGG GCC target DNA target DNA CCG GGCTTAA 3' 5' r Cloning of PCR products - I —► attachment of restriction sites and restriction cleavage GAATTC ^^^^^J ATGC AGGTAG GCTAGTGTCA H CGATCACAGT NNNCG amplification t restriction cleavage you can use use PCR to make lots of copies of the insert something with insert you want and you can use the primers to add on cut sites you want (TO 4 this cutting leaves you with phosphorylated ends but the primers are usually synthesized without phosphates - this only comes into play if your vector is dephosphorylated https://thebumblingbiochemist.com/365-days-of-science/molecular-cloning-using-restriction-enzymes/ Fcioning of PCR products - II TA cloning Taq polymerase creates single-nucleotide 3'-overhangs, most often A ligation CCT| GGQ WLaz-Prom KJAG - pCRII-TOPO NLaz-Prom [TTC - DH5a™ Escherichia coli D. Perez Torres et al. / Revista Clinica (2011) r Introduction the construct into the host Transformation = introduction of non-viral DNA into prokaryotes and non-animal eukaryotes Transfection = introduction of DNA into a euraryotic cell Transduction = transfer of DNA using viral vectors Methods of transformation/transfection • Heat shock + CaCI • Electroporation • Lipofection • Microinjection and „gene gun" „Heat Chemocompetent cells with CaCI2 shock" Add DNA, 4° C, 30 min DNA & the cell membrane are both negatively-charged so they'd normally repel Calcium ions treatment DNA lipopolysaccharides Ca' lets them stick Allow me to bring yoii\ guys some positivity! — 2+ + Ca2+ helps the DNA get "in place" Negative charges on DNA Temperature treatment .OOoo 0.0 V % 0Q S Y o heat shock opens the , pores wider and \ helps some of the Q ' * DNA get in Hi https://thebumblingbiochemist.com/365-days-of science/bacterial-transformation-heat-shock- chemically-competent-cells/ DNA tnnittmů into the cell https://doi.Org/10.3389/fmicb.2017.02169 Selection of transformants Electroporation Electrical impulses cause the formation of pores in the cell membrane and the entry of exogenous DNA into the cell Before Pulse During E-field After Pulse Cell membrane Introduce genes/drugs Electric field induces a voltage across cell membrane Cell "heals" with gene/drug inside + n ■ https://www.socmucimm.org/news-media/bacterial-transformation-electroporation^ r Lipofection Structure of the synthetic cationic lipid component of the TransFast™ Reagent. Formation of liposomes with encapsulated DNA Possibility of transfection of oligo DNA, RNA, siRNA, YAC Widely used in eukaryotes Possibility of transfection in vivo DNA Lipoplex Moghaddam, B. (2013). Design and development of cationic liposomes as DNA vaccine adjuvants. *—+ Cationic Liposome Cell Membrane Endosomal Release Figure 1.5: Proposed mechanisms of cationic lipoplex condensation and uptake. In brief, cationic liposomes are attracted by electrostatic interactions to the negative charges of DNA forming a lipoplex. Lipoplex binding to the cell surface followed by intemalisation and then release of DNA from the lipoplex. DNA enters the nucleus and in the nucleus, RNA will be transcribed. „Gene gun" and microinjection " * https://doi.org/10.1016/C2011-0-05817-9 Mechanical introduction of DNA into the cell - mainly suitable for eukaryotes due to the size of the "vectors". Microinjection - introduction of DNA directly into the nucleus of a cell (eggs, embryonic stem cells...) "Gene gun" (bioballistic technique) - injecting nano-/microparticles coated with DNA into cells Target plant cells Selection of cells with a recombinant gene 1) Restriction analysis of plasmid DNA after mini-preparation 2) Inactivation by the insert 3) a-complementation 4) Hybridization of colonies 5) PCR test 6) Sequencing f Selection is primarily based on antibiotic resistance Cloning foreign DNA using E. coli, cont. Cloning site The host cell is sensitive to the antibiotic The vector carries resistance genes /i-.-.HI Oniiin of replication (on) null The transformed cell is resistant to the antibiotic Determine colonies with insert using selection on antibiotic plates https://library.uams.edu/assets/COM/BioChem/Molecul arTools/MolecularToolsSDLIO.html Host DNA i Transformation of E. coli cells All colonies have plasmids Colonies with recombinant plasmids Al selection of transformed cells Agar containing tetracycline colonies transferred for testing Agar containing Agar containing tetracycline (control) ampicillin + tetracycline nactivation by the insert - TETS selection I EcoR\,Xap\ 4359 >>3fll 4284 Ss/7l 4168 fiSUlSi 23 Mm! Ill 29 Pst\ 3607 VSp\ 3537. ECB31I 3433 f*m1105l 3361 ,fcu32l 185 Nhei 229 BamHI 375 Pael 562 Xag\ 622 -^Ss/I 651 BstM 1063 £co52l 939 68I 972 Ca/l 2884 /M,flhJ-o_ < oh OH ho oh oh 7 HO 0 Allolactose oh Galactose HO OH Glucose [ * * j Lac Repressor with Allolactose r~i Lac Repressor bound to Operator Dl o z m mm Induction of Lac Operon Glycolysis 1 (7G ATP/Lactose) J J Multidis Res Rev 2018 Figure 1: Diagram summarizing the functions of p-galactosidase in the cell. The enzyme can hydrolyze lactose to galactose plus glucose, it can transgalactosylate to form allolactose, and it can hydrolyze allolactose. The synthesis of allolactose which binds to the lac repressor and reduces its affinity for the lac operon is as a result of the presence of lactose. This in tum allows the synthesis of p"-galactosidase, the product of the laeZ gene. Operon of Escherichia coli enzymes for lactose metabolism Regulatory gene lac operon Promoter DNA Pf lacl gene Plgc 0 lacZ gene lacY gene lac A gene } Operator /acAmRNA /ac-mRNA I I Repressor J protein /3-galactosidase Galactoside permease Trans-acetylase S 2012 Pearson Education, Inc. I: code represor Z: code enzyme beta-galactosidase (lac -> glu + gal) Y: code enzyme permease A: code enzyme thiogalactoidtransacetylase inductor - laktose Regulation of lac Operon a) glc is NOT present, lac is NOT present binding of the repressor to the operator b) glc is NOT present, lac IS present -> enzyme induction c) glc IS present, lac IS present catabolic represion r Regulation of lac operon 1 Glucose HIGH Lactose LOW RNA polymerase bound to lad promoter Glucose HIGH Lactose HIGH RNA polymerase bound to lad promoter Glucose LOW Lactose HIGH RNA polymerase bound to lad promoter Figure 17-10 Biological Science, 2/e Operator RNA polymerase bound loosely to promoter NOTRANSCRIPTION lacZ lacY lack CAP site RNA polymerase bound ~\m loosely to promoter INFREQUENT TRANSCRIPTION lacZ lacY lacA RNA polymerase bound tightly to promoter FREQUENT TRANSCRIPTION lacZ lacY lack © 2005 Pearson Prentice Hall, Inc. Regulation of ß-galactosidase expression I polylinker (MCS) IPTG Isopropyl ß-D-1 -thiogalactopyranoside operator lacZ another genes represor IPTG i ch20h ohJ—o s- oh The medium must not contain glucose! Formation of blue coloration HQ |_0 OH H20 [3-Galactosidase X-Gal Spontaneous Br dimerization and oxidation 5,5 -Dibromo-4,4 -di grows on bacteria in the form of plaques > it only needs about 2/3 of the genome to infect > up to 20 kbp of DNA can be cloned > 78-105% length DNA can be packaged efficiently Hybridization of colonies nylon membrane colony imprint on the membrane _ lysis, denaturation fixation bacterial colony (genome bank) chromosomal DNA fingerprint Addition of denatured labeled cDNA íl X-ray film colony identification exposition overnight incubation, hybridization ^Testing of recombinant plasmids by the | PCR method 1 vector 1 cloning site vector vector primers for sequencing Confirmation of the presence of the insert using primers for sequencing primer 1 vector I vector primer 2 primer 1 vector Insert with length X vector primer 2 Determining the orientation of the insert in the vector sequencing primer 1 primer forward -► -► vector Insert with length x vector <- 4 primer reverse sequencing primer 2 Amplicons are created by combining primers > sequencing primer 1 + sequencing primer 2 (amplicon length = N + X) > forward primer + reverse primer (amplicon length = X) > sequencing primer 1 + reverse primer > sequencing primer 2 + forward primer sequencing primer 1 ssj9A9j jeuiud -► -► vector x MlBuai mum uasui vector A- A- pjeywjoj jeuiud sequencing primer 2 Sequencing > decisive method > each insert needs to be sequenced A C G T w. •> * = .5 - > 1 'aaagc ctggggtgcctaatg 180 190 agtga gc ta ac tcacatta 200 210 at tgc gttgc gc tcac tocccgct' 220 230 240 rTCCAGTCGGG a a AC ctgtc gtgccagc tgcattaatga atcggc ( 250 260 270 280 :aac gcgcgggga gaggc gc 290 300 t ttgc gtattgggc gctc ttcc gc ttcc tcgc tcac tgac tc gc tgc gc tc g gtc gttc ggc tg< 310 320 330 340 . 350 360 37c