Lecture 7 4-12

• Cellular engineering is the application of engineering tools and concepts to the study and manipulation of living cells.
• Cellular engineering is necessarily interdisciplinary, combining tools and ideas from the fields of biology, engineering, applied mathematics, physics, and chemistry.
• Genetic engineering, also known as genetic modification, is the process of altering the DNA in a genome.
• Genetic engineering may involve changing one base pair (A-T or C-G), deleting a whole region of DNA, or introducing an additional copy of a gene.
• Genetic engineering is used by scientists to enhance or modify the characteristics of an individual cell/organisms.
• Applications of genetic engineering

  Genetic engineering of cells have several important applications one of them being metabolic engineering
• Metabolic engineering

  Practice of optimizing genetic and regulatory processes within cells to increase the cell's production of a certain substance. These processes are chemical networks that use a series of biochemical reactions and enzymes that allow cells to convert raw materials into molecules necessary for the cell's survival.
• Cancer Immunotherapy (CAR T-cells)

  Cancer immunotherapy is the use of the immune system to treat cancer.
• Gene Expression describes the flow of genetic information in cells from DNA to messenger RNA (mRNA) to protein. It states that genes specify the sequence of mRNA molecules, which in turn specify the sequence of proteins.
• Transcription = synthesis of an RNA that is complementary to one of the strands of DNA
• Translation = ribosomes read a messenger RNA and make protein according to its instruction
• Information from the genes is transferred to a unique RNA sequence which in turn specifies the sequence of proteins. Three important processes in the cell:

  • DNA replication: biological process of producing two identical replicas of DNA from one original DNA molecule
  • Transcription: synthesis of an RNA that is complementary to one of the strands of DNA. This RNA, called a messenger RNA, is then read by ribosomes which convert this sequence to a protein during a process called translation.
  • Translation: ribosomes read a messenger RNA and make protein according to its instruction
• Gene expression in prokaryotes

  Bacteria continuously need to adapt to their environment, so controlling the expression of genes into proteins is very important. Bacteria have developed various ways to control how much protein is being expressed. Three different levels of control:

  • Transcriptional control
  • Translational control
  • Post-translational control
• Transcriptional control = bacteria can control the rate at which transcription can take place. By controlling this rate they are able to make more or less mRNA which results in an increased or decreased level of proteins.
• Translational control = regulating translation of RNA into protein. For example bacteria can change the translational rate as well as the life time of messenger RNA.
• Post-translational control = activating or inhibiting proteins using small molecules or targeted degradation.
• An operon is a functional complex of genes containing the information for enzymes of a metabolic pathway. It includes:

  • Operator - site next to the promotor, where the regulatory protein can bind
  • Regulator gene - encodes for transcription factors (repressor)
  • Promotor - where the RNA polymerase binds
  • Structural genes - encodes functional enzymes and are translated from a single mRNA (polycistronic)
• Lac operon = negative inducile operon (usually OFF: needs signal to turn ON)

  • structural genes encode enzymes that metabolize lactose
  • No lactose: bacteria don't need to make the enzymes to metabolize lactose -> the repressor binds to the lac operator and represses transcription of genes
  • Lactose present: bacteria want to metabolize it -> allo(lactose) binds the repressor -> released from operator -> transcription of the genes is turned on
• trp operon = repressible operon (usually ON; needs signal to turn OFF)

  • Codes for a regulatory protein and several enzymes that produce tryptophan
  • No tryptophan: bacteria need to make it -> gene expression ON
  • Tryptophan present: bacteria don't need to make it -> repressor binds tryptophan -> binds operator -> gene expression OFF
• Gene expression in prokaryotes

  • Negative inducible and repressible operons -> make use of repressor = negative gene regulation
  • Positive gene regulation -> via a stimulatory activator protein

  Example: Catabolite activator protein (CAP) lac operon.

  Glucose low -> cAMP high -> cAMP binds CAP -> induce gene expression -> increase in enzymes that can convert lactose to glucose
• Post-transcriptional control

  Previous examples were all based on control over the rate of transcription initiation as these transcription factor control binding of RNA polymerases to the promoter. However, bacteria can also control the rate of transcription termination as well as the rate of translation using various different types of mechanisms such as attenuation (transcription termination) and riboswitches (transcription termination & translation initiation)
• Riboswitches

  Bacterial mRNA transcript contains couple of regions: (1) coding region (starts with start codon and ends with stop codon), this is the region that is translated into a protein. (2) 5' untranslated region (UTR) and (3) 3' UTR.
• Riboswitches are RNA elements that are incorporated in the 5' untranslated region of bacterial messenger RNA.
• The 5' untranslated region is the place before the protein-coding region beginning with the start codon (AUG) and ending with a stop codon (UAA).
• After the stop codon the 3' untranslated region begins.
• The 5' UTR also contains the shine-dalgarno sequence (= ribosome binding site) which helps recruit the ribosome to the mRNA to initiate protein synthesis by aligning the ribosome with the start codon.
• Riboswitches that are present in the 5' untranslated region adopt different secondary structures to effect gene regulation depending on whether ligand is bound.
• Riboswitches can bind small molecules and thereby influence the rate of translation.
• Prokaryotic genome = one circular chromosome, accessible, transcription and translation occurs in same space

  Eukaryotic genome = multiple chromosomes, DNA packed into chromatin, transcription and translation physically separated
• Eukaryotes vs prokaryotes

  • Expression of genes in eukaryotes more complex than prokaryotes
  • Prokaryotes contain a single circular chromosome -> very accessible. In addition, transcription and translation occurs in the same compartment.
  • In eukaryotic cells, DNA organized in multiple chromomeres and DNA packed into chromatin
• Gene expression in eukaryotic cells is regulated at various levels including chromatin modifications, DNA methylation, histone acetylation, and control of transcription (enhancers).
• Chromatin modifications refer to changes in the local structure of chromatin which can increase or decrease transcription.
• DNA methylation is a process where methyl groups are added to DNA, which can alter gene expression.
• Histone acetylation is a process where histone proteins are modified by the addition of acetyl groups, which can affect gene expression.
• Control of transcription (enhancers) involves transcription factors binding to specific sites on DNA and can inhibit or activate the expression of a certain gene.
• Alternative splicing, which occurs at the RNA level, is a process where micro RNA in eukaryotic cells undergo splicing and this splicing can be regulated.
• Degradation of mRNA, or messenger RNAs, can be actively regulated, for example, using microRNA.
• Blockage of translation, or the translation of mRNA into proteins, can also be positively or negatively regulated.
• Protein degradation is a process where proteins can be actively targeted for degradation.