RECOMBINANT DNA TECHNIQUES and PROTEIN ENGINEERING

1. RECOMBINANT DNA TECHNIQUESandPROTEIN ENGINEERING Vectors

2. Plasmids prokaryotic eukaryotic Phage M13 lambda Cosmids Artificial chromosomes Cloning vectors Expression vectors Vectors

3. Plasmids • First plasmid described was found in Japan in Shigella species during an outbreak of dysentery in the early 1940's • Three plasmid types are the most studied: F factor (fertility factor), R plasmids (antibiotic resistance) and Col (colicinogenic) • R plasmids - medically importantPenicillin was introduced for general use in early 1940's1946 - 14% of Staphylococcus aureus were penicillin resistant1947 - 38% PenR1969 - 59% PenR1970's - almost 100% of S. aureus were PenR • F factor: important for pilus formation

4. 4 groups of plasmids based on transfer properties: • Nontransmissible - can neither initiate contact with recipient nor transfer DNA • Conjugative - can initiate contact with recipient bacterium • Mobilizable - can prepare its DNA for transfer • Self-transmissible - is both conjugative and mobilizable • Donation - a conjugative plasmid (such as F) can provide conjugative function to a mobilizable plasmid (such as ColE1) such that both plasmids can be transferred. • Plasmid conduction - a self-transmissible plasmid (such as F) can recombine with a non-mobilizable plasmid and transfer the cointegrate.

5. Plasmid replication - by theta replication (either uni- or bidirectional) or rolling circle • Replicon - DNA molecules that can replicate autonomously (plasmids, chromosomes, phage) • Replicon must have on origin of replication • Plasmid origin of replication called oriV (ori vector) • Functions of the ori region • Host range - narrow or broad host ranges • Copy number determination

6. Regulation of the copy number at the ColE1 origin of replication • RNAse H cuts DNA-RNA hybride • Moderately high copy number (~ 50 / cell) • Elimination/mutation of rop gene increases copy number (>200 / cell)

7. pBR322: first successful cloning vector (Bolivar et al. 1977) (4363 bp) rep/ori pMB1 plasmid Apr (bla) Tn3 transpozon TcrpSC101 plasmid

9. Selection I • Selection for the uptake of plasmid • Antibiotic resistance • Ampicillin • Tertacycline • Chloramphenicol • Streptomycine • Kanamycine

10. Antibiotic resistance genes ampicillin cell wall synthesis-laktamase (karbenocillin) tetracyclineaa-tRNA binding40 kD membrane protein chloramfenikol peptidyl-transferasechl-acetyltransferase (CAT) streptomycin initiation , missreadingst.-phosphotransferase kanamycin missreading kan.-acetyltransferase (neomycin)

11. Selection II • Selection for the presence of insert in the plasmid • pBR322: insertional inactivation, replica plating • pUC: „blue-white” selection

12. pUC18/19 (Vieira & Messing, 1982) (2686 bp) pMB1 (ColE1) replikon „MCS” (polylinker) (57 bp, 10 sites) (18/19 ) reverse orientation lacZ’ (CAP binding site Plac, operator, RBS, -gal -fragment: 60 aa, MCS: instead of codon no. 6 and 7

13. lac OPERON

14. a complementation: „blue – white” selection

15. M13 phage • Single stranded, filamentous phage • Does not lyse the cells • 10, non overlaping genes • Double stranded form in the cell (RF) • Rolling circle replication generates single stranded

16. Life cycle of M13 phage Infection through the pilus Assembly in the membrane

18. pBluescript (phagemid) • 21 restriciton sites (in two orientations) • Blue-white selection • Promoters for RNA pol and sequencing • f1 ori in two orientations • single stranded rescue • needs helper phage! • Restiction sites with alternating 3’ and 5’ overhang • Nested deletions

20. pTR262 PR (-phage) promoter – tetracycline resistence; cI gene insert in cI gene pZErO (Invitrogen) insert in ccdB killer gene cytotoxic F plasmid gene (DNA gyrase toxine) gyrA462 strain is resistant (to clone vector) cI Pr Tc Positive selection vectors (e.g.)

21. VECTORS for GRAM POSITIVE BACTERIUMS • Bacillus subtilis, Staphylococcus aureus • Streptomycetes • plasmid and phage based vectors “SHUTTLE” VECTORS • Two replicons • e.g. E. coli / B. subtilis; E. coli / yeast or mammalian

22. -PHAGE VECTORS temperated bacteriophage (icosahedral head + flexible tail) • linear doublestranded genome (48,5 kb, ~50 gén) • lysogen (prophage) vs. lytic lifecycle (cI, cro, cII, cIII, N, Q) • adsorption (maltose transporter LamB), circularization (cos sites), - and „rolling-circle” replication, packaging; integration and induction • genomic és cDNS libraries • 38 kb < recombinant  > 52 kb (78%-105%) • left arm (A-J virion genes;~20 kb)right arm (pL, pR, regulation, ori; ~10 kb) • + insert concatamer

25. Phage  vectors • Insertion • Substitution • Ligation • concatamers • in vitro Packaging • Gigapack(Strategene) • terminase, head and tail proteins • infection: 107-109 pfu/mg

26. Why is there a LOWER size limit? • A molecular motor spins DNS intocapsidhead • The pressure must be 15-30 atm

27. INSERTIONS VECTORS: (cDNAlibraries) lgt10 (“immunity” vector) • cloning site: cI434 gene(EcoRI) • insert: 0-7 kb • selection: repressor inactivationscreening: probe hybridization lgt11 • cloning site: lac-Z gene(EcoRI) • selection: blue-white(IPTG + X-Gal) • screening: probe hybridization. lacZ fusion proteinantibody

28. -PHAGE VECTORS • ZAP FAMILY(Stratagene) (Short et al., 1988) • in vivoexcision of inserts( in form of pBluescript) • helper filamentous phage(e.g. ExAssist) • gene II amber mutation+ SupE host(XL1-Blue) • phagemid, helper, ZAP(thermal inactivation) • Sup0host(SOLR) pBluescript

29. COSMIDS plasmid - -phage hybrid vectors (~8 kb) 1 v. 2 cos-site (250 bp) pMB1 replicon in vitro packing, infection,plasmid replication insert: 30-45 kb genomic libraries, genomemapping (YACsubclones) Lawrist4, pWE15, SuperCos (Stratagene) FOSMID: F plasmid replicon (1-2 copy)

30. P1-PHAGEVECTORS P1 (temperate) bacteriophage(110 kb) • insert: 75-100 kb • in vitro packing, • „headful” mechanism; pac site (162 bp), packase enzym, phage head and tail • infection, site specific recombination(linearphage circular plasmid) • cre/loxP system( P1 prophage hostcre recombinase) • plasmid replication • P1 plasmid (1 copy) és lytic replicon (multiple copies)

31. The cre-lox system Cre recombinase The Cre (Cyclization Recombination) protein consists of 4 subunits and two domains  : The larger carboxyl (C-terminal) domain, and smaller amino (N-terminal) domain. The total protein has 341 amino acids. The C domain is similar in structure to the domain in the Integrase family of enzymes isolated from Bacteriophage λ. This is also the catalytic site of the enzyme. Lox P site Lox P (locus of X-over P1) is a site on the Bateriophage P1 consisting of 34 bp. There exists an asymmetric 8 bp sequence in between with 2 sets of palindromic, 13 bp sequences flanking it.

32. pAd10-SacBII (30 kb) 2 vector domain betweenloxP sites - ColE1 replicon, ampr, pac site, stuffer (11 kb) - P1 plasmid Rep, lyt Rep (+ lac operon), kanr, E. coli sacB gene + MCS positive selection (levane synthetase)

34. The cre-lox system Cre recombinase

35. ARTIFICIAL CHROMOSOMES • stable, no recombination, (vs.. YAC clones) • BacPac Resources (http://bacpac.med.buffalo.edu/), GenomeSystemsInc. • PAC • (P1-derived Artificial Chromosome) • electroporation • pCYPAC2, pPAC4 (19 kb) • BAC • (Bacterial Artificial Chromosome) • insert: >300 kb • F faktor replikon (oriS, repE, parA,B) (F „miniplasmid”) • elektroporation • pBAC108L, pBACe3.6 • Cmr, cosN, loxP • YAC (Yeast artificial chomosomes) [yeast vectors]