CEN 551: Biochemical Engineering

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CEN 551 Biochemical Engineering

• Instructor Dr. Christine Kelly
• Chapter 8

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Schedule

• Exam 2. Thursday, March 4, before spring break.
• Exam 2 Take home on chapter 8 material,
  in-class on Chapter 9, 10 and 11.
• Take home exam - Genetic engineering.
• In-class - operation of bioreactors, scale-up and
  control of bioreactors, recovery and
  purification.
• 3 weeks from Thursday.

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Homework

• By Friday at 500 pm email ckelly_at_syr.edu a 1
  paragraph description of your project topic.
• Chapter 8
• Problems 2, 6, 7, and 8.
• Due Thursday, February 19.

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Chapter 8 How cellular information is altered

• Mutation and Selection
• Natural Mechanisms for Gene Transfer and
  Rearrangement
• Genetically Engineering Cells
• Genomics

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• We can alter cells by using mutation or genetic
  engineering. Mutation is subjecting the cells to
  stress causing changes in the genetic make-up.
  Genetic engineering is the purposeful transfer of
  DNA from one type of organism to another.

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Mutations and Selection

• Mutations mistakes in the genetic code (can
  arise from replication and/or damage)
• Mutant organism with a genetic mutation
• Wild type the organism without the genetic
  organism
• Genotype genetic construction of an organism
• Phenotype characteristics expressed by an
  organisms.
• Expression usually refers to transcriptiontrans
  lationposttranslation processing

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Examples

• Strain A has the tol operon for toluene
  degradation, and is in a reactor growing on
  glucose.
• Strain B has the tol operon for toluene
  degradation, and is in a reactor growing on
  toluene.
• These strains have the same genotype, but
  different phenotypes.

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Point mutation single base change

• Consequences base change may or may not result
  in an amino acid change.
• If the amino acid is the same as before the
  mutation there is no consequence.
• If the amino acid is different, but not in the
  region of the active site, there may be no
  consequences.
• If the mutation is in the active site, there may
  be some enzyme activity consequence.
• If the mutation changes the amino acid to a stop
  codon, the resulting protein will be truncated
  and probably not active.

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Selection

• Selectable mutation confers upon the mutant an
  advantage for growth, survival or detection under
  a set of environmental conditions that the wild
  type does not have.
• Examples
• Antibiotic resistance
• Ability to grow on toluene
• Inability to produce lysine
• Ability to produce bioluminescence
• Ability to produce more of an enzyme
• Inability to grow at higher temperatures

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Serial Dilution Plating
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Natural Mutation Rates

• 10-3-10-9 mutations per cell conversion
• 10-6 1 mutation/1,000,000 divisions
• How do we increase mutation rates?
• Why do we want to increase mutation rates?

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• Increase Mutation Rates
• Mutagens chemicals, radiation
• Lots of growth (i.e. lots of divisions)

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Why do we want to increase mutations?

• We want a cell to develop specific
  characteristics that are advantages for us.
• For example, removing feed back inhibition of
  lysine to increase lysine production

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Natural Gene Transfer/Rearrangement

• Transformation uptake of free DNA by a cell.
  The cell membrane has to be permeable to DNA.
• Transduction DNA is carried into the call in a
  phage.
• Conjugation Cell to cell transfer of DNA. Also
  called mating.
• Once the DNA is inside the cell it can remain
  separate from the chromosome in self replicating
  plasmid, or integrate into the chromosome. To
  integrate, the DNA must be complementary to the
  chromosomal DNA on the ends.

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Mutation and Selection

• Using mutation and selection engineers and
  microbiologists were able to increase penicillin
  from 0.001 g/L to 50 g/L.

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Genetic Engineering

• Using natural mechanisms to purposefully
  manipulate DNA. The DNA is manipulated outside
  of the cell, and then sent into the cell.

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Genetic Engineering Tools

• Restriction enzymes enzymes that cut DNA at
  specific sequences. Different enzymes will cut
  at different sequences.
• Gel electrophoresis (Southern Blot) A method to
  detect what sizes of DNA a sample contains.
• Polymerase chain reaction (PCR) A process used
  to make many copies of a piece of DNA.
• Plasmid self replicating, circular piece of DNA
  that can survive in a cell.

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http//www.accessexcellence.org/AB/GG/nucleic.html
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Gel Electrophoresis
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Polymerase Chain Reaction
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PCR Primers DNA polymerase nucleotides
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Polymerase Chain Reaction (PCR)

• PCR allows scientists to extract and analyze
  bits of microbial DNA from samples, meaning they
  dont need to find and grow whole cells. PCR is
  an essential element in DNA fingerprinting and in
  the sequencing of genes and entire genomes.
  Basically, its like a technique to photocopy
  pieces of DNA. In a matter of a few hours, a
  single DNA sequence can be amplified to millions
  of copies. PCR lets scientists work with samples
  containing even very small starting amounts of
  DNA.

http//www.microbeworld.org/htm/aboutmicro/tools/g
enetic.htm
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• The technique makes use of the DNA repair enzyme
  polymerase. This enzyme, present in all living
  things, fixes breaks or mismatched nucleotides in
  the double-stranded DNA helix. These breaks or
  mismatches could cause genes to malfunction if
  left unfixed.

http//www.microbeworld.org/htm/aboutmicro/tools/g
enetic.htm
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• Polymerase uses the intact half of the DNA
  molecule as a template and attaches the right
  nucleotides, which circulate constantly in the
  cell, to the complementary nucleotide at the site
  of the break. (DNA consists of two strands of
  nucleotide bases, which are represented as A, G,
  C, and T. In the laws of DNA base-pairing, A
  joins with T and G with C.)

http//www.microbeworld.org/htm/aboutmicro/tools/g
enetic.htm
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• Not all polymerases are created equal, however.
  Many fall apart in high heat. PCR was developed
  in 1985 following the discovery of an unusual
  heat-loving bacterium called Thermus aquaticus in
  a hot spring in Yellowstone National Park. This
  bacteriums polymerase, dubbed Taq, does its job
  of matching and attaching nucleotides even in the
  high heat generated by the successive
  photocopying cycles required during PCR. Taq
  made PCR possible.

http//www.microbeworld.org/htm/aboutmicro/tools/g
enetic.htm
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http//www.microbeworld.org/htm/aboutmicro/tools/g
enetic.htm
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Plasmids and Cloning
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Movies

• 65601 missense mutation
• 65701 nonsense mutation
• 95301 bacterial transformation
• 153301 mutation
• 151401 virulence transformation
• 156401 heat DNA
• 92201 restriction enzyme, recombination
• 112601 PCR
• 165401 Sequencing

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Example

• I have two organisms 1. A fast growing yeast
  that grows well in a fermentor. 2. A fungi that
  is difficulty to grow.
• The fungi produces an enzyme that may be
  valuable, but I cannot grow enough fungi to
  produce enough enzyme to even test the enzyme.
• How can I use the genetic engineering tools to
  get enough enzyme?

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Genetic Engineering

1. PCR the enzyme DNA from the fungi, get a bunch of
   the DNA that encodes for the valuable enzyme.
2. Find a restriction enzyme that will cut the
   valuable enzyme DNA on ether side (but not in the
   middle).
3. Obtain a plasmid that will replicate in the
   yeast, that has a site that the same restriction
   enzyme will cut downstream of a strong promoter.
4. Cut the valuable enzyme DNA and the plasmid with
   the restriction enzyme.
5. But the valuable enzyme DNA and plasmid together
   and let them recombine.
6. Get the plasmid into the yeast.
7. At all steps, use gel electrophoresis to check
   and make sure you have the right DNA.

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White-Rot Fungi

• Fungi with mycelium type growth.
• Able to degrade lignocellulosic materials using
  several enzyme systems (lignin and manganese
  peroxidases, laccases).
• Expresses and secretes MnP under nitrogen
  limitation at low concentrations.
• Expresses several degradative enzymes has been
  widely studied for bioremediation applications.
• Not suitable for conventional industrial
  fermentations.

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White-rot Fungi P. chrysosporium
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Manganese Peroxidase

• Glycosylated enzyme that uses H2O2 to oxidize
  manganese, which in turn oxidizes lignin.
• White-rot fungi produces a 41-47 kDa MnP under
  secondary metabolism.
• Native fungal secretion signal directs secretion
  out of the cell.
• Requires a heme cofactor for ligninolytic
  activity.

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glycosylation
MnP crystal structure. (Sundaramoorthy et al.,
1994).
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Pichia pastoris

• Methylotrophic (methanol as a sole carbon source)
  yeast.
• Capable of eukaryotic post translational
  modifications.
• Higher yields, less expensive, higher cell
  density, and easier to scale up than mammalian
  and fungal systems.
• Secretes only small amounts of native proteins.
• Many cloning and expression vectors available.

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Efforts to Increase Production of MnP

• Homologous expression
• P. chrysosporium primary metabolism low
  concentration
• Heterologous expression
• Bacteria (E. coli) inactive inclusion bodies
• Insect cells active enzyme, low concentration (5
  mg/L), heme addition, expensive
• Fungal (Aspergillus spp.) active enzyme, higher
  concentration (100 mg/L), heme addition

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Cloning of mnp

• 1. The white-rot fungi was grown under nitrogen
  limitation.
• 2. The total RNA was extracted from the
  culture.
• Reverse transcriptase polymerase chain reaction
  (RT-PCR) was performed with oligo dT primers to
  create DNA complementary to the mRNA.
• PCR was performed with primers specific for the
  MnP gene.

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tubes
Thermocycler (PCR machine)
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• 4. PCR was perfomed again with restriction
  enzyme sites built into the primer sequence. So
  the ends of the MnP gene would have the correct
  restriction sites.
• 5. The PCR product was cut with restriction
  enzymes.
• 6. E. coli with the pGAP vector was grown, and
  then the pGAP vector was isolated from the
  culture. The pGAP vector was cut with the same
  restriction enzymes.

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• 6. The PCR product (a tube full of MnP gene DNA)
  was mixed with the pGAP vector and the enzyme
  ligase. The sticky ends of the MnP gene were
  complementary to the sticky ends of the pGAP
  vector, and they hybridized. The ligase enzyme
  will then ligated the nucleotides together. The
  result was a tube with some pGAP vector (dimers)
  with no MnP insert, some MnP gene DNA (dimers),
  and some pGAP vector with an MnP insert.

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• 7. This mixture of DNA was placed in a cuvette
  with live P. pastoris cells, and an electric
  current passed through the solution.

cuvette
electroporator
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• 8. The electric current caused the membrane to
  be permeable to DNA. The DNA in the solution
  went into the P. pastoris cells.
• 9. Samples from the tube of P. pastoris cells
  were plated to selective medium with the
  antibiotic zeocin. The pGAP vector has
  antibiotic resistance genes. The cells that grew
  into visible colonies had the pGAP vector insert.
  These colonies were grown, and tested to check
  if the cells produced MnP using an enzyme
  activity method.

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Pichia pGAP vectorconstitutive promoter
affinity
antigen
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mnp1 cDNA Sequencing Result (fungal secretion
signal, 2 mutations) TCAGCTCTCAAGGACATCCGCACTCGAA
TATCGCA ATGGCCTTCGGTTCTCTCCTCGCCTTCGTGGCTCTCGCCGCC
ATAACTCGCGCCGCCCCGACTGCGGAGTCTGCAGTCTGTCCAGACGGTAC
CCGCGTCACCAACGCGGCGTGCTGCGCTTTCATTCCGCTCGCACAGGATT
TGCAAGAGACTCTGTTCCAGGGTGACTGTGGCGAAGATGCCCACGAAGTC
ATCCGTCTGACCTTCCACGACGCTATTGCAATCTCCCAGAGCCTAGGTCC
TCAGGCTGGCGGCGGTGCTGACGGCTCCATGCTGCACTTCCCGACAATCG
AGCCCAACTTCTCCGCCAACAGCGGCATCGATGACTCCGTCAACAACTTG
CTTCCCTTCATGCAGAAACACGACACCATCAGTGCCGCCGATCTTGTACA
GTTCGCCGGTGCGGTCGCGCTGAGCAACTGCCCAGGTGCTCCTCGCCTCG
AGTTCATGGCTGGACGTCCGAACACTACCATCCCCGCAGTTGAGGGCCTC
ATTCCTGAGCCTCAAGACAGCGTCACCAAAATCCTGCAGCGCTTCGAGGA
CGCCGGCAACTTCTCGCCGTTCGAGGTCGTCTCGCTCCTGGCTTCACACA
CCGTTGCTCGTGCGGACAAGGTCGACGAGACCATCGATGCTGCGCCCTTC
GACTCGACACCCTTCACCTTCGACACCCAGGTGTTCCTCGAGGTCCTGCT
CAAGGGCACAGGCTTCCCGGGCTCGAACAACAACACCGGCGAGGTGATGT
CGCCGCTCCCACTCGGCAGCGGCAGCGACACGGGCGAGATGCGCCTGCAG
TCCGACTTTGCGCTCGCGCGCGACGAGCGCACGGCGTGCTTCTGGCAGTC
GTTCGTCAACGAGCAGGAGTTCATGGCGGCGAGCTTCAAGGCCGCGATGG
CGAAGCTTGCGATCCTCGGCCACAGCCGCAGCAGCCTCATTGACTGCAGC
GACGTCGTCCCCGTCCCGAAGCCCGCCGTCAACAAGCCCGCGACGTTCCC
CGCGACGAAGGGCCCCAAGGACCTCGACACGCTCACGTGCAAGGCCCTCA
AGTTCCCGACGCTGACCTCCGACCCCGGTGCTACCGAGACCCTCATCCCC
CACTGCTCCAACGGCGGCATGTCCTGCCCTGGTGTTCAGTTCGATGGCCC
TGCCTAA  
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Expression of rMnP1
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 Antibody based slot blot detection to identify
rMnP protein in the supernatant of recombinant P.
pastoris culture
Fungi Positive control
Recombinant P. pastoris
No insert
medium
blank
Recombinant P. pastoris
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Western Blot rMnP1 Detection (various media and
concentration methods)
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Intracellular Expression of rMnP
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Glycosylation

• Three potential N-glycosylation sites.
• Native MnP only glycosylated at one site as
  indicated by crystal structure.

Asparagine
Serine/threonine
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Effect of Deglycosylation on rMnP
native MnP
rMnP
untreated
untreated
degly1
degly2
degly1
degly2
150 kDa
degly1 PNGaseF
degly2EndoH
45 kDa
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Western Blot Time Course rMnP
fungi
Recombinant P. pastoris
time
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Problem 8.3. Isolate a temperature sensitive
mutant able to grow at 30oC but not at 37oC.

1. Expose cells to mutagen (uv light, chemicals).
2. Dilution plate the culture to obtain a plate with
   widely separated colonies.
3. Replicate plate.
4. Place one plate at 30oC and the other at 37oC.
5. Select the colony that grew at 30oC but not at
   37oC.

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• Expose cells to mutagen (uv light, chemicals).

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Serial Dilution Plating
2. Dilution plate the culture to obtain a plate
with widely separated colonies.
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3. Replicate Plating
Can produce plates with identical strains in the
same location.
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4. and 5. This strain grew at 30oC, but not at
37oC.
30oC
37oC
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HW Problem 8.2

• Obtain a methionine overproducer.
• The primary objective is to mutate enzyme r so
  the allosteric inhibitor site is no longer
  functional in other words, remove the feed back
  inhibition by methionine.
• Subject the culture to a mutagen chemical or
  radiation.
• Plate the mutagenized culture on an appropriate
  medium with a dilution sufficient to allow
  individual colonies, widely separated.
• Screen the colonies for methionine over
  production.

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HW Problem 8.6

• Given the amino acid sequence of the peptide,
  what is the sequence of steps to obtain an E.
  coli strain expression the peptide.
• Reverse translate the amino acid sequence to
  obtain a DNA sequence that will encode for the
  peptide.
• Design end sequences to create a restriction site
  at each end of the peptide DNA.
• Chemically synthesize the DNA.

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• Design primers for the the synthesized DNA.
• PCR using the synthesized DNA as a template.
• Cut the PCR product DNA with the restriction
  enzyme.
• Cut an appropriate vector with the same
  restriction enzyme.
• Mix the cut PCR product with the cut vector with
  the enzyme ligase.
• Transform (electroporation or mating) the vector
  into the E. coli cell.
• Plate the transformed cells on selective medium
  with the antibiotic that the vector carries
  resistance to.

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• Test the colonies that form for expression of the
  peptide using protein gel electrophoresis with
  antibody staining.

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HW Problem 8.7

• A protein converts colorless substrate to blue
  product. We want to produce and E. coli
  expressing this protein. We have a high-copy
  plasmid with penicillin-resistance.
• We cannot chemically synthesize the DNA because
  it is too large (protein not a peptide). We must
  find donor DNA.
• We can screen many organisms for the ability to
  turn the colorless substrate the blue. We will
  use on that can as the source DNA to encode for
  the protein.

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• We can design primers based on the beginning and
  ending of the protein amino acid sequence. We
  will need a set of primers because the redundancy
  of codons that encode for a particular amino
  acid.
• We can PCR the genomic DNA of or source strain
  using different combinations of the primers.
• Run the PCR product on a gel and search for the
  size band the same as the DNA that encodes for
  the protein. That set of primers was correct for
  the ending sequence.
• Design new primers to incorporate restriction
  sites at the ends of the gene and PCR the source
  DNA.
• Cut both the vector DNA and the PRC product with
  the appropriate restriction enzyme.

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• Combine the cut vector, the cut DNA and the
  enzyme ligase to obtain a vector with the gene
  insert.
• The vector will have either an inducible or
  constitutive promoter.
• Transform the vector with the insert into the E.
  coli. And plate the transformed E. coli onto
  penicillin medium with the colorless substrate.
• Select the colonies that appear and are blue.

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HW Problem 8.8

• Similar problem 8.6. The vector should have an
  inducible or constitutive promoter. The vector
  or the gene should have transcription and
  translation stop codons.

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Clarification, Review, and Take Home Exam

• Assume we have the DNA sequence of a protein.
  This is the sequence of the coding strand.
• The non-coding strand is complementary to the
  coding strand.
• The mRNA is synthesized from the non-coding
  strand making the sequence of the mRNA the same
  as the sequence for the DNA coding strand (except
  for the Us).
• The translation start on the mRNA is AUG, so the
  translation start on the coding DNA strand is
  ATG. There needs to be some mRNA transcribed
  before the translation start for the mRNA to bind
  to the ribosome.

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• The translation stop codons are the nonsense
  codons indicated in table 4.1 in your text. The
  most common is UAG.
• The transcription start and stop sites are not as
  simple as a single codon. They are defined by
  the promoter and terminator regions which are
  typically on the vector already. It is OK if the
  mRNA is longer than the corresponding protein
  it has to be longer at the front end. The extra
  sequences are not translated.

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Take Home Exam

• We would like to produce an enzyme in a
  recombinant host because the native host is not
  suitable for bioreactor fermentation.
• The enzyme is to be used for a transformation in
  commodity scale food process.
• Suggest the steps for creating the recombinant
  strain.

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• Select the recombinant host E. coli, yeast, or
  CHO cells. Briefly explain why you selected the
  host.
• We know the coding strand DNA sequence. The
  flanking sequences, including the start and stop
  are indicated below.
• AGATCTCTGGUUATGGAATTC..AAGCTTCGAUAGGATTAGATCT
• If you use PCR give the sequence of the primers
  you will use.
• If you use restriction enzymes, name which
  restriction enzyme you will use.
• Use diagrams with sequence information whenever
  possible explaining your steps.

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Assume you have a vector for each type of host
with the indicated elements.
Strong promoter
Promoter and antibiotic resistance gene
Origin of replication