Engineering magnetosomes to express novel proteinsWhich ones? •Must be suitable for expressing in Magnetospyrillum!•Can’t rely on glycosylation, disulphide bonds, lipidation, selective proteolysis, etc for function!• Best bets are bacterial proteins• Alternatives are eukaryotic proteins that don’t need

any of the above• Short peptides

•Tweaking p18• Linker• Deleting or replacing GFP

•TRZN•Oxalate decarboxylases•Lactate dehydrogenase or other oxalate metab enzyme

How to bioengineer a novel protein expression system?1.Identify a suitable candidate organism2.DNA must function in this host3.mRNA must function in this host4.Protein must be functional•Must be able to purify large amounts of functional protein

How to bioengineer a novel protein expression systemNew host must be able to “read” the sequencePromotersTerminatorsCapping, splicing and polyA if eukaryoteCodon usage

How to bioengineer a novel protein expression systemNew host must be able to “read” the sequencePromotersTerminatorsCapping, splicing and polyA if eukaryoteCodon usage3. mRNA must function in the new host

How to bioengineer a novel protein expression system?3. mRNA must function in the new host•Must be translated: needs correct ribosome-binding site

How to bioengineer a novel protein expression system?3. mRNA must function in the new host•Must be translated: needs correct ribosome-binding site• S-D in prok• Kozak in euk

How to bioengineer a novel protein expression system?3. mRNA must function in the new host•Must be translated: needs correct ribosome-binding site• S-D in prok• Kozak in euk• Also needs correct information in 5’ UTR

How to bioengineer a novel protein expression system?3. mRNA must function in the new host•Must be translated: needs correct ribosome-binding site• Also needs correct information in 5’ UTR• In eukaryotes also needs 5’ cap & poly-A tail

How to bioengineer a novel protein expression system?3. mRNA must function in the new host•Must be translated: needs correct ribosome-binding site• Also needs correct information in 5’ UTR• In eukaryotes also needs 5’ cap & poly-A tail• Must also be properly localized

How to bioengineer a novel protein expression system?3. mRNA must function in the new host•Must be translated: needs correct ribosome-binding site•Must survive: in euk depends on internal sequences (especially in 3’ UTR) and poly-A tail

How to bioengineer a novel protein expression system?4. Protein must function in the new host•Must be translated•Must be modified correctly

How to bioengineer a novel protein expression system?4. Protein must function in the new host•Must be translated•Must be modified correctly• Glycosylation: affects folding, solubility &

localization

How to bioengineer a novel protein expression system?4. Protein must function in the new host•Must be translated•Must be modified correctly• Glycosylation: affects folding, solubility &

localization• Simple glycosylation can be engineered

Protein must function in the new hostMust be modified correctlyGlycosylationLipidation•Myristylation (14C) @ N•Palmitoylation (16C)•Isoprenoids

Protein must function in the new host•Must be modified correctly• Glycosylation• Lipidation• proteolysis

Protein must function in the new host•Must be modified correctly• Glycosylation• Lipidation• Proteolysis• Disulfide bonds

Protein must function in the new host•Must be modified correctly• Glycosylation• Lipidation• Proteolysis• Disulfide bonds

Must go to correct location

Protein must function in the new host•Must be modified correctly• Glycosylation• Lipidation• proteolysis

Must go to correct locationMust survive!

How to bioengineer a novel protein expression system?3. Protein must function in the new host• Must be modified correctly• Glycosylation• Lipidation• proteolysis

• Must go to correct location• Must survive!

4. Identify suitable gene(s), then obtain in some way, add suitable promoters and terminators, and transform into new host.

How to bioengineer a novel protein expression system?Hosts?•Escherichia coli

• Pro• Best-understood & fastest growing of all hosts• Most genetic control• Lots of tricks & special-purpose strains

How to bioengineer a novel protein expression system?Hosts?•Escherichia coli

• Pro• Best-understood & fastest growing of all hosts• Most genetic control• Lots of tricks & special-purpose strains

• Con• Prokaryote: no glycosylation, S-S bonds, etc.

How to bioengineer a novel protein expression system?Hosts?•Escherichia coli

• Pro• Best-understood & fastest growing of all hosts• Most genetic control• Lots of tricks & special-purpose strains

• Con• Prokaryote: no glycosylation, S-S bonds, etc.

•Saccharomyces cerevisiae (brewer’s yeast)• Pro• Eukaryotic• Fast-growing and well-characterized• Good genetic control• Lots of tricks & special-purpose strains

Hosts?E. coliS. cerevisiae (brewer’s yeast)• Pro• Eukaryotic• Fast-growing and well-characterized• Good genetic control• Lots of tricks & special-purpose strains

• Con• Does things its own way• Weird glycosylation, etc• Proteins from other eukaryotes often don’t work

when expressed in yeast

Hosts?•E. coli•Saccharomyces cerevisiae (brewer’s yeast)•Pichia pastoris

• Pro• Methylotrophic: grows in [methanol] that kill

most other organisms• Eukaryotic• Fast-growing and well-characterized• Good genetic control with glucose & MeOH• Grows to very high densities

Hosts?•E. coli•Saccharomyces cerevisiae (brewer’s yeast)•Pichia pastoris

• Pro• Methylotrophic: grows in [methanol] that kill

most other organisms• Eukaryotic• Fast-growing and well-characterized• Good genetic control with glucose & MeOH• Grows to very high densities

• Con• Does things its own way• Weird glycosylation, etc• Proteins from other euk often don’t work

Hosts?•E. coli•Saccharomyces cerevisiae (brewer’s yeast)•Pichia pastoris•Baculovirus in cultured insect cells• Pro• Make glycoproteins “faithfully”• Can’t infect plants or animals• Can use promoters of “late genes” to drive

expression of heterologous proteins• Insect cell cultures are easier & more forgiving

than mammalian cell cultures

Hosts?•E. coli•Saccharomyces cerevisiae (brewer’s yeast)•Pichia pastoris•Baculovirus in cultured insect cells• Pro• Make glycoproteins “faithfully”• Can’t infect plants or animals• Can use promoters of “late genes” to drive

expression of heterologous proteins• Insect cell cultures are easier & more forgiving

than mammalian cell cultures• Con• Trickier• Requires cell cultures: expensive!

Hosts?•E. coli•Saccharomyces cerevisiae (brewer’s yeast)•Pichia pastoris•Baculovirus in cultured insect cells•Transgenic plants

• Pro• Make authentic plant proteins (and sometimes

animal proteins)• Cheap• “Easy” and robust techniques

Hosts?•E. coli•Saccharomyces cerevisiae (brewer’s yeast)•Pichia pastoris•Baculovirus in cultured insect cells•Transgenic plants

• Pro• Make authentic plant proteins (and sometimes

animal proteins)• Cheap• “Easy” and robust techniques

• Con• Slow• May not process non-plant proteins correctly

Hosts?•E. coli•Saccharomyces cerevisiae (brewer’s yeast)•Pichia pastoris•Baculovirus in cultured insect cells•Transgenic plants•Animal cell cultures

• Pro• Make authentic animal proteins• “Easy” and robust techniques

Hosts?•E. coli•Saccharomyces cerevisiae (brewer’s yeast)•Pichia pastoris•Baculovirus in cultured insect cells•Transgenic plants•Animal cell cultures

• Pro• Make authentic animal proteins• “Easy” and robust techniques

• Con• Slow• Expensive!• Purifying proteins can be difficult

Hosts?•E. coli•Saccharomyces cerevisiae (brewer’s yeast)•Pichia pastoris•Baculovirus in cultured insect cells•Transgenic plants•Animal cell cultures•Transgenic animals

• Pro• Make authentic animal proteins

Hosts?•E. coli•Saccharomyces cerevisiae (brewer’s yeast)•Pichia pastoris•Baculovirus in cultured insect cells•Transgenic plants•Animal cell cultures•Transgenic animals

• Pro• Make authentic animal proteins

• Con• Slow• Expensive!• Difficult• Purifying proteins can be difficult

Hosts?Magnetospirillum gryphiswaldense

Hosts?Magnetospirillum gryphiswaldense•Can propagate plasmids (but pBAM requires pir gene)•Or can insert into chromosome via tnpA (Tn5)-based transposition: no variation in expression due to copy number or growth stage

Hosts?Magnetospirillum gryphiswaldense•Borg optimised rbs, promoter & codon usage

Hosts?Magnetospirillum gryphiswaldense•Borg optimised rbs, promoter & codon usage•Developed inducible system based on tetracycline

Hosts?Magnetospirillum gryphiswaldense•Borg optimised rbs, promoter & codon usage•Developed inducible system based on tetracycline•Fuse protein to C-terminus of mamC

Hosts?Magnetospirillum gryphiswaldense•Borg optimised rbs, promoter & codon usage•Developed inducible system based on tetracycline•Fuse protein to mamC C-terminus: exposed at surface

Hosts?Magnetospirillum gryphiswaldense•Borg optimised rbs, promoter & codon usage•Developed inducible system based on tetracycline•Fuse protein to mamC C-terminus: exposed at surface•Purify with magnets

Assignment•Design a mamC C-terminal protein fusion

• Design DNA sequence encoding a useful protein

Assignment•Design a mamC C-terminal protein fusion•Design DNA sequence encoding a useful protein•Replace eGFP of pJH3 with your protein

• Best to use MluI and NheI sites

AssignmentBest to use MluI and NheI sitesDesign oligos that add MluI in frame at 5” end and NheI at 3’end

AssignmentBest to use MluI and NheI sitesDesign oligos that add MluI in frame at 5’ and NheI at 3’endDigest vector & clone with MluI and NheI then ligate

AssignmentBest to use MluI and NheI sitesDesign oligos that add MluI in frame at 5’ and NheI at 3’endDigest vector & clone with MluI and NheI then ligateFind & analyze clones

Transcription

Prokaryotes have one RNA polymerase

makes all RNA

core polymerase = complex of 5 subunits (’)

Transcription

Prokaryotes have one RNA polymerase

makes all RNA

core polymerase = complex of 5 subunits (’)

not absolutely needed, but cells lacking are very sick

Initiating transcription in Prokaryotes1) Core RNA polymerase is promiscuous

Initiating transcription in Prokaryotes1) Core RNA polymerase is promiscuous2) sigma factors provide specificity

Initiating transcription in Prokaryotes1) Core RNA polymerase is promiscuous2) sigma factors provide specificity• Bind promoters

Initiating transcription in Prokaryotes1) Core RNA polymerase is promiscuous2) sigma factors provide specificity• Bind promoters• Different sigmas bind different promoters

Initiating transcription in Prokaryotes1) Core RNA polymerase is promiscuous2) sigma factors provide specificity• Bind promoters3) Once bound, RNA polymerase “melts” the DNA

Initiating transcription in Prokaryotes3) Once bound, RNA polymerase “melts” the DNA4) rNTPs bind template

Initiating transcription in Prokaryotes3) Once bound, RNA polymerase “melts” the DNA4) rNTPs bind template5) RNA polymerase catalyzes phosphodiester

bonds, melts and unwinds template

Initiating transcription in Prokaryotes3) Once bound, RNA polymerase “melts” the DNA4) rNTPs bind template5) RNA polymerase catalyzes phosphodiester

bonds, melts and unwinds template6) sigma falls off after ~10 bases are added

Structure of Prokaryotic promotersThree DNA sequences (core regions)

1) Pribnow box at -10 (10 bp 5’ to transcription start)5’-TATAAT-3’ determines exact start site: bound by factor

Structure of Prokaryotic promotersThree DNA sequences (core regions)

1) Pribnow box at -10 (10 bp 5’ to transcription start)5’-TATAAT-3’ determines exact start site: bound by factor

2)” -35 region” : 5’-TTGACA-3’ : bound by factor

Structure of Prokaryotic promotersThree DNA sequences (core regions)

1) Pribnow box at -10 (10 bp 5’ to transcription start)5’-TATAAT-3’ determines exact start site: bound by factor

2)” -35 region” : 5’-TTGACA-3’ : bound by factor3) UP element : -57: bound by factor

Structure of Prokaryotic promotersThree DNA sequences (core regions)

1) Pribnow box at -10 (10 bp 5’ to transcription start)5’-TATAAT-3’ determines exact start site: bound by factor

2)” -35 region” : 5’-TTGACA-3’ : bound by factor3) UP element : -57: bound by factor

Structure of Prokaryotic promotersThree DNA sequences (core regions)

1) Pribnow box at -10 (10 bp 5’ to transcription start)5’-TATAAT-3’ determines exact start site: bound by factor

2)” -35 region” : 5’-TTGACA-3’ : bound by factor3) UP element : -57: bound by factorOther sequences also often influence transcription! Eg CAP site in lac promoter

Termination of transcription in prokaryotes

1) Sometimes go until ribosomes fall too far behind

Termination of transcription in prokaryotes

1) Sometimes go until ribosomes fall too far behind

2) ~50% of E.coli genes require a termination factor called “rho”

Termination of transcription in prokaryotes

1) Sometimes go until ribosomes fall too far behind

2) ~50% of E.coli genes require a termination factor called “rho”

3) Our terminator (rrnB) first forms an RNA hairpin, followed by an 8 base sequence TATCTGTT that halts transcription