Manipulation of gene Expression in Bacteria

1. Manipulation of gene Expression in Bacteria EXPRESSION EXPRESSION EXPRESSION

2. Biotechnology • The primary objective of gene cloning or genetic engineering is to express the cloned gene in a selected host organism

3. Host System • Bacteria • Yeast • Insect • Mammalian cells

4. GENETICALLY ENGINEERED PRODUCTS • Some products produced by genetic engineering and are currently on the market • Bt potatoes • PRV Papaya • Herbicide resistant corn • Human growth hormone • Insulin • human interferon protein • monoclonal antibodies

5. Gene expression in different systems • Bacterial genes being expressed in a eukaryotic system • Eukaryotic genes are being expressed in bacteria

6. Expression vectors • Vectors designed to overproduce specific gene products • Use well-characterised RNA and protein synthesis systems • Lambda ZAPII • pTrc99A • pBluescript II SK+

7. Manipulations to modulate gene expression • The transcriptional promoter and terminator sequences • The ribosome-binding site and the efficiency of translation in the host organism • The number of copies of the cloned gene and whether the gene is plasmid borne or integrated into the genome of the host • The intrinsic stability of the cloned gene protein within the cell • The final cellular location of the synthesized foreign protein

8. Promoters • Effective gene expression system requires a strong and regulatable promoter sequence upstream from the cloned gene • Strong promoter has a high affinity for RNA pol

9. Regulated promoter • Some cloned DNA may produce products that are toxic to the bacterial cell, and if the promoter driving production of such gene products were not regulated, the bacterial cell might be poisoned. • High levels of continual expression of a cloned gene is often detrimental to the host cell because it creates an energy drain, thereby impairing essential host cell functions. • Plasmids carrying a constitutively expressed gene may be lost after several generation since cells without plasmids can take over the culture

10. What is the best promoter type for You? • Constitutive promotersalways express your gene of interest and eliminate the extra complexity of adding an inducer. • If you have a non-toxic gene and are not worried about the timing of your expression, using a constitutive promoter is easier.

11. Examples of Promoters • T3 • T5 • T7 • Lac • Trp • 35S from CMV • Chicken  actin

12. Promoter… • The 35S promoter for CMV is used to express viral genes in plants because a bacterial promoter or bacteriophage promoter cannot turn on genes in plants • Mammalian expression uses the strong constitutive CAG promoter that consists of the chicken  actin promoter

13. Tri Systems www.Qiagen.com • A single vector is possible in three different expression systems • the T5 promoter/lac operator transcription-translation system for expression in E. coli • the p10 promoter for baculovirus-based expression in insect cells • the CAG (actin) promoter for expression in mammalian cells

14. Regulated expression vector pTrc99A (Ref: Amann et al. Gene 69: 301-315 (1988)) • Designed for the regulated expression of genes in E. coli of fused and non-fused proteins • Based on pKK233-2 • Origin of replication from pBR322 • Has a strong hybrid promoter trp/lac • The lacZ ribosome binding site (RBS) • MCS of pUC18 • rrnB transcription terminator

16. Ptac or Ptrc promoter • Regulated hybrid promoter containing the –35 promoter from the trp promoter and the –10 region from the lacuv5 promoter • Lacuv5 is a variant of the lac promoter that contains altered nt seq in the –10 region is stronger than the wild type lac promoter and is repressed by the lac repressor and derepressed by IPTG or lactose • Does not require activation with CAP-cAMP

17. Lac • In the absence of lactose, the E. coli lac promoter is repressed, turned off by the lac repressor protein • Induction can be achieved by the addition of lactose or IPTG, prevents the binding of the repressor to the lac operator • Catabolite activator protein (CAP) increases affinity of the promoter for RNA pol

18. Trp • Trp promoter is negatively regulated, turned off by a tryptophan trp-repressor protein complex that binds to the trp operator and prevents transcription of the trp operon • De repression, or turning on achieved by removing tryptophan or adding 3-indole acrylic acid

19. Consensus sequences • 5’-TATAAT-3’ for the –10 region • 5’-TTGACA-3’ for the –35 region • The lacUV5 promoter has a consensus sequence at its –10 but not its –35 region • The trp promoter has a consensus sequence at its –35 region but not its –10 region • By fusing the –10 region of the lac promoter and the –35 region of the trp promoter, called it the Ptac or Ptrc

20. pTrc99A • lacI gene including its corresponding promoter (PlacI). •  Also carries the lacIq mutation for which helps to maintain repression of Plac transcription. • Produces much higher levels of the lac repressor, thereby decreasing the leakiness under non-induced conditions, i.e. transcription of a cloned gene in the absence of an inducer

21. pTrc99A • To produce the protein in pTrc99A, cells carrying the plasmid are grown to a moderate density and IPTG is added

22. Increasing protein production • Strategy used to increase protein production in recombinant E. coli involves designing an expression vector with a Ts origin of replication and a regulated promoter • Replacing the origin of replication of the plasmid pPLc2833 with that from the plasmid pKN402 • To make the plasmid pCP3 • pCP3 contains the pL promoter, the -lactamase gene for amp resistance from pLc2833 and the origin of replication from pKN402

24. cI repressor is present in the chromosome of the E. coli host • The pL promoter is controlled by the cI repressor protein of the bacteriophage lambda, this switches off the transcription from pL promoter • Cells that carry the pCP3 plasmid are grown first at 28C and then shifted to 42C • At 28C the cI repressor is functional and turns off the pL promoter plasmid copy number is normal • At 42C, the plasmid copy number increases and the ts cI repressor is inactivated

25. Large scale production systems • Temperature shifts take time and energy • Chemical inducer e.g. IPTG is expensive

26. Large scale production systems • Two Plasmid system: • In one plasmid: • The cI repressor was placed under the trp promoter (Ptrp) and inserted into a low copy plasmid • In a second plasmid: • Have the cloned genes, e.g. -galactosidase and citrate synthase genes under the pL promoter

28. Termination signals • Two strong transcription terminators – T7 from E. coli bacteriophage T7 and rabbit globin terminator sequence • Contains signals for the polyadenylation of the mRNA transcript – to prevent read-through transcription to ensure stability of the expressed construct

29. Regulatory sequences

30. Ribosome binding site (RBS) • The molecular basis for differential translation is the presence of a translational initiation signal called a ribosome binding site (rbs) in the transcribed mRNA

31. RBS • Bacterial cells and human cells have different mechanisms for selecting translational start points on the mRMA • This is an important consideration for producing human proteins in bacteria

32. RBS- Bacteria • Directed by a ribosome attachment sequence closely linked to the AUG initiator codon for translation. • This ribosome attachment sequence consist of 6 – 8 nucleotides UAAGGAGG (Shine Dalgarno sequence) upstream of AUG • This sequence is complementary to the 3’ end of the 16S ribosomal RNA, AUUCCUCC

33. RBS • Activity of a RBS can be influenced by the length and nucleotide composition of the spacer separating the RBS and the initiator AUG • Bacterial mRNAs that do not have a close match to the consensus ribosome attachment sequence are not translated efficiently • Generally, the stronger the binding of the mRNA to the ribosomal RNA, the greater the efficiency of translational initiation

34. RBS - Eukaryotes • The ribosome binds to an mRNA through the 5’ terminal cap structure • The ribosome then scans the RNA in the 5’ to 3’ direction until it located the first AUG in the mRNA • If this AUG is surrounded by a suitable sequence, translation will begin

35. RBS - Eukaryotes • This sequence in eukaryotes, called the Kozak sequence 5’-A/GCCACCAUGG which lies within a short 5' untranslated region, directs translation of mRNA

36. Conditions required for maximum translation efficiency • The RBS must be located at a precise distance from the translation start codon of the cloned gene • Overproducing a human protein in E. coli requires customizing the gene to have a bacterial RBS appropriately spaced from the AUG codon for translation initiation • The DNA sequence that includes the RBS through the first few codons of the gene must not contain nucleotide sequences that after transcription can fold back to form intra-strand loops, thereby blocking the interaction of the mRNA with the ribosome.

37. Expression vector pKK233-2 • An ampicillin resistance gene • The tac promoter • The lacZ ribosome binding site • An ATG start codon located 8 nucleotides downstream from the RBS • The transcription terminators T1 and T2 from lambda

39. Copies of the cloned gene • Tandem gene arrays • level of gene expression is proportional to the number of copies of the transcribed gene in the host cell

40. STRATEGY • Increase plasmid copy in the cell • Clone multiple copies of the gene in tandem into a low copy number plasmid

41. Vector Preparation • Unique site in vector is digested with AvaI (C_TCGGG) • Filling in with DNA polymerase I • EcoRI linker (GAATT_C) inserted • EcoRI linker flanked by two AvaI sites (CTCGGAATTCTCGGG) is inserted into the plasmid • The gene plus the signals are cloned into the EcoRI site by digesting with AvaI to give non-sticky ends so the genes are orientated in one direction

43. DNA integration into the host chromosome • When a gene is part of the host chromosomal DNA, it is relatively stable and consequently can be maintained for many generations

44. Factors to consider when integrating a gene • the chromosome integration site must not be within an essential coding gene • the input gene must be under the control of a regulated promoter • for integration the DNA sequence must have some sequence homology for recombination between the two DNA molecules • chromosomal site about 50 nt

46. Integration into the host chromosome • The cloned gene inserted in the middle of a cloned segment of DNA (ab) from the host chromosome on the plasmid • Homologous DNA pairing occurs between plasmid-bourne DNA regions a and b and the host chromosome DNA regions a’ and b’ • A double cross over event (X-X) results in the integration of the cloned gene

48. Stability of cloned gene protein • Small proteins and peptides are difficult to be stably expressed in E. coli • These proteins can be stabilised by expressing them fused to a large protein such as mouse (DHFR) dihydrofolate reductase

49. Fusion Proteins • Fusion proteins are constructed at the DNA level by ligating together the coding regions of two genes • One gene is the host gene that produces a stable host protein and the other gene represents the cloned gene that will produce a foreign protein • When the two genes are transcribed, the foreign protein produced will be covalently linked to the stable host protein

50. Uses of fusion protein • determine its location in a cell • stability of the protein • used as antigens and to generate antibodies • Used to purify recombinant proteins through the technique of immuno-affinity chromatography