1. High-Throughput Cloning and Expression Library Creation for Functional Proteomics The International Proteomics Tutorial Program

2. What is cloning? DNA cloningOrganism cloning Cloning is a process where a genetically identical copy of an organism, cell or DNA is produced Dolly sheep

3. DNA cloning and functional proteomics DNA Functional proteomics Cell based assays Cell proliferation Morphology Drug resistance Protein function Interaction partners PTMs Gene of interest Clone Protein expression in vivo or in vitro

4. Expression Libraries Traditional Library DNA Clone Library Expression library is the collection of clones capable to express protein. It stores genetic information to facilitate the study of the roles of genes and proteins in a systematic and high-throughput (HTP) manner.

5. Main enzymes used for DNA cloning (1)DNA polymerases (2)Restriction enzymes and ligases (3)Recombinases

6. DNA polymerase DNA polymerase can use RNA or DNA as template. DNA dependent-DNA polymerase copy the DNA and can be used for DNA amplification in vitro in a Polymerase Chain Reaction (PCR)

7. Polymerase Chain Reaction

8. DNA polymerase DNA polymerase can use RNA or DNA as template. DNA dependent-DNA polymerase copy the DNA and can be used for DNA amplification in vitro in a Polymerase Chain Reaction (PCR) RNA dependent-DNA polymerase is used to copy the information from the RNA to the DNA. The new DNA molecule is name copy DNA or cDNA

9. cDNA synthesis

10. Type II Restriction Enzyme and Ligase Type II Restriction enzymes – “scissors” capable of identifying and cleaving specific DNA sequences Ligase - re-join two DNA strands

11. Recombinases Recombinases can cut and ligate DNA fragments, within a single complex Bidirectional recombination

12. Recombinases Unidirectional recombination Recombinases can cut and ligate DNA fragments, within a single complex

13. Steps for DNA cloning 1)Identification of appropriate insert and insert source 2)Selection of appropriate vector 3)Selection of appropriate cloning strategy

14. Insert selection - What is the structure of the gene in the genome? Prokaryotic RNA 5’UTR Coding sequence(CDS) StartStop 3’UTR Open Reading Frame (ORF) Prokaryotic genomic DNA Coding sequence(CDS) Transcription

15. Eukaryotic genomic DNA Mature eukaryotic mRNA Pre-mRNA 5’ 3’ Cap 5’UTR Coding sequence(CDS) 3’UTR Poly-A tail StartStop 3’ Capping, splicing, polyadenylation Open Reading Frame (ORF) 5’ 5’UTR 3’UTR Exon Intron Exon 5’ 5’UTR 3’UTR Exon Intron Exon Insert selection - What is the structure of the gene in the genome?

16. ORFs – The insert of choice for expression clones Due to the lack of introns, ORFs can be expressed to study protein functions. Templates to Generate ORF Clones (1)Genomic DNA: all genes equally present but not suitable for genes with introns (most eukaryotes); (2) cDNA: no introns between exons but the biases on mRNA expression exist both temporary and spatially; (3) Vector with the gene of interest (GOI): gene specific primers can be designed to amplify the GOI for the cloning into the desired vectors; (4) Gene Synthesis (Chemical or PCR-based method). Allows the codon usage optimization. High cost.

17. Templates to generate ORF clones

18. Vectors Entry vector common features: ori (origin of replication) selection marker cloning site Inserts cloned into entry vectors are NOT expressed

19. Expression vectors Additional features in the expression vector : promoter (required) tag (optional)

20. Necessary characteristics of high-throughput cloning systems Properties of the enzymes used for cloning Fidelity Efficiency Stability Clone validation Automation Friendly Cost

21. Example of available high- throughput cloning systems 1.Restriction Enzyme Based Cloning (1)Flexi® Vector Systems (Promega) 2.Recombination Based Cloning (1) Creator Cloning System (Cre-loxP, Clontech) (2) Gateway® Technology (phage lambda – attB/P, Life Technologies)

22. Flexi® vector systems Flexi cloning system is a restriction enzyme-based cloning system that uses two rare-cutting restriction enzymes, SgfI and PmeI Flexi vectors carry a lethal gene in between SgfI and PmeI to allow the efficient cloning selection. Frequency of cDNAs or open reading frames(ORFs) lacking SgfI or PmeI sites Flexi® Vector Systems Technical Manual@Promega

23. Creating Flexi® clones Lethal gene in the expression vectors provides negative selections for the parent vectors

24. Generation of Flexi clones with amino-terminal tag 5’…GTTTAAAC…3’ 3’…CAAATTTG…5’ PmeI sequence: Reconstitution of SgfI site encodes Ala-Ile-Ala and PmeI site encodes Val-Stop-Thr. GOI will be translated from the tag’s start codon to the stop codon present in the gene or the PmeI site (in red); SgfI sequence: 5’…GCGATCGC…3’ 3’…CGCTAGCG…5’ + Digestion, ligation and selection

25. 5’…GTTTAAAC…3’ 3’…CAAATTTG…5’ 5’…GAGCTC…3’ 3’…CTCGAG…5’ PmeI EcolCRI 5’…GTTTCTCNN…3’ 3’…CAAAGAGNN…5’ New Sequence Ligation Ligation of PmeI and EcolCRI sites disrupt both PmeI and EcolCRI sites, new sequence encodes Val-Phe-X, X determined by GNN; It is inadvisable to transfer GOI into C-terminus Flexi vectors if you plan to transfer it into other Flexi vectors in the future. Generation of Flexi clones with carboxyl-terminal tag + Digestion, ligation and selection

26. Transferring genes between distinct Flexi vectors Acceptor vector with amino-terminal tag SgfI & PmeI digestion Ligation Transformation Antibiotic 2 selection

27. SgfI & PmeI digestion SgfI & EcolCRI digestion Ligation Transformation Antibiotic 2 selection Transferring genes between distinct Flexi vectors Acceptor vector with carboxyl-terminal tag + +

28. Creator cloning system Creating Master Clones With In-Fusion: In-Fusion enzyme fuses DNA fragments, e.g., PCR generated sequences and linearized vectors, precisely by recognizing a 15bp overlap at their ends. Creating Expression Clones With Creator Cloning: 1. Cre recombinase mediates the recombination between DNA sequences at loxP sites; 2. Donor vectors contain two loxP sites, which flank the 5’ of GOI and 5’ of CmR resistance gene; 3. Acceptor vectors contain a single loxP site, followed by a bacteria promoter to facilitate the expression Cm R gene after recombination. LoxP sequences: In-Fusion® HD Cloning Kit User Manual@Clontech

29. Creating entry clones with In-Fusion

30. Creating expression clones with Creator cloning system

31. Tags in the Creator expression vectors N-terminal Tag

32. Tags in the Creator expression vectors C-terminal Tag

33. Gateway cloning system Creating Gateway Master Clones Using BP reactions BP clonase recombine linear PCR product with entry vectors. Creating Gateway Expression Clones Using LR reactions LR clonase recombine entry clone with expression vectors.

34. 5’UTR 3’UTR Gene of Interest(GOI) Generation of PCR fragment with attB sites Gene of Interest(GOI) 1 st PCR using gene specific primers + Partial attB1/attB2 site sequences 2 nd PCR using universal primers including the full length attB1/attB2 site sequences attB1attB2 Gene of Interest(GOI) attB1/attB2 site sequences are adapted from phage λ that allows the site specific recombination between DNA fragments; Two rounds of PCR are applied for the addition of adaptor sequences to avoid potential mutations in the synthesis of long primers.

35. Creating Gateway entry clones using BP reactions

36. Creating Gateway expression clones using LR reactions

37. Tags in the Gateway expression vectors N-terminal Tag

38. Tags in the Gateway expression vectors C-terminal Tag

39. Generating new expression vectors Flexi Cassette Gateway Cassette Creator Cassette Overall principle: Insert the required cloning cassette into the desired expression vectors by any cloning method (restriction enzyme, In-Fusion).

40. Alternative cloning methods In-Fusion

41. Alternative cloning methods Gibson Assembly

42. Alternative cloning methods Ligation Independent Cloning

43. Alternative cloning methods Gold Gate Assembly

44. Alternative cloning methods Univector Plasmid Fusion System

45. Comparison of distinct cloning systems

47. Proteomics application of expression clone libraries Generation of protein microarray with 2000 distinct ORFs from Vibrio cholerae for the identification of immune response in patients with Cholera Adapted from Festa et al, 2012 DNA Protein