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INF > INTRO TO MICROBIAL BIOTECHNOLOGY > Flashcards

INTRO TO MICROBIAL BIOTECHNOLOGY Flashcards

1
Q

ORIGIN OF THE WORD ‘ENZYME’?

A
  • DERIVES FROM GREEK FOR ‘LEAVENED’ (meaning in dictionary: (of bread) made with yeast or other raising agent)

History:
- MICROBIAL TECHNOLOGY ISN’T NEW –> YEAST THOUGHT TO BE USED IN BAKING/BREWING FOR AT LEAST 5,000 YEARS
- MICROORGANISM (YEAST) IDENTIFIED AS RESPONSIBLE FOR BREAD RISING IN THE 1800s (LOUIS PASTEUR)
(helps understand underlying meaning of the word enzyme)

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2
Q

WHEN DID MODERN MICROBIAL BIOTECHNOLOGY HIT A STEP-CHANGE? WHAT ENABLED IT?

A
  • INT EH 1960s-70s WITH THE MAINSTREAMING OF MOLECULAR CLONING
    + CONTRIBUTING FACTORS: DISCOVERY OF PCR AND RESTRICTIVE ENZYMES
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3
Q

WHAT WAS THE 1ST RECOMBINANT THERAPEUTIC (Recombinant therapeutics are therapeutic proteins produced by recombinant DNA technology) DEVELOPED IN A MICROBIAL SYSTEM? WHEN?

A

HUMAN INSULIN, 1977

‘Recombinant human insulin’ became available in large amounts by recombinant DNA technology using fermentation in microorganisms (bacteria or yeast). Recombinant insulin has a superior level of purity and consistent quality compared with semisynthetic insulin.

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4
Q

ESTIMATED VALUE OF THE ENTIRE MICROBIAL TECHNOLOGY MARKET IN 2022

A

307,500,000,000$

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5
Q

PROPORTION OF CLINICLLY APPROVED THERAPEUTICS DERIVED FROM NATURAL PRODUCTS?

A

CCA 70%

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6
Q

NUMBER OF PEOPLE WORDLWIDE TAKING STATINS + FUNCTION OF STATINS + ORIGIN?

A

200 000 000 PEOPLE WORLDWIDE

  • STATINS ARE CHOLESTEROL LOWERING DRUGS
  • A FUNGAL NATURAL PRODUCT
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7
Q

WHAT IS BIOMASS?

A

A RENEWABLE ENERGY SOURCE GENERATED FROM ANIMAL OR PLANT MATERIALS

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8
Q

WHICH ‘ENVIRONEMENT’ IS THE LARGEST POSSIBLE SOURCE OF BIOMASS?

A

MARINE SEDIMENT

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9
Q

WHICH ‘ENVIRONEMENT’ IS THE MOST CULTURED BUT THE SMALLEST SOURCE OF BIOMASS? WHY IS IT EXTENSIVELY CULTURED THEN?

A

ANIMALS (ANIMAL HOSTS)

THERE IS BIAS TOWARDS STUDYING ANIMALS CAUSE MOST MICROBES THAT CAUSE DISASE COME FROM THEM

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10
Q

HOW TO FURTHER INVESTIGATE ORGANISMS WHICH CANNOT BE CULTURED OR ARE VERY CHALLENGING TO CULTURE? DESCRIBE THE PROCESS

A
  • RELYING ON GENOMICS
  • TRY TO IDENTIFY AS MUCH AS POSSIBLE OF THE ORGANISM’S GENOME AND LOOK FOR SPECIFIC AREAS OF INTEREST (SEQUENCING, ESP METAGENOMICS SEQUENCING)
  • RELEVANT GENE CLUSTER IS OFTEN MOVED INTO A MORE COOPERATIVE ORGANISM (LIKE SOME BACTERIAS) THAT IS MORE EASILY CULTURED, EASIER TO SCALE, PURIFY THE MATERIAL ETC
  • THE SYSTEM PRODUCES NUMEROUS BIOACTIVE COMPOUNDS THEN, AND THEY ARE SCREENED TO SEE IF THERE IS ANYTHING OF INTEREST
  • AFTER OVERCOMING THE ISSUE OF CULTURING, WHAT IS CONSIDERED NEXT IS HOW TO EXTRACT THE USEFUL COMPOUNDS
  • MAINLY A CONCERN OF ANALYTICAL CHEMISTRY
  • SEPARATION SCIENCES (CHROMATOGRAPHY), STRUCTURAL CHARACTERISATION (NMR, MASS SPECTROMETRY), HIGH OUTPUT (OFTEN AUTOMATED) ACTIVITY SCREENS OF FRACTIONATED MIXTURES
  • EVEN WHEN CULTURING AND ISOLATION ARE COMPLETED, PROBLEMS MIGHT PRESENT WITH SCALE (OPTIONS: AGAIN, MOVE TO A COOPERATIVE BUG THAT IS KNOWN TO SCALE WELL OR PASS OVER TO MEDICINAL CHEMISTS TO TRY AND MAKE THE COMPOUND SYTHESIS)
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11
Q

METAGENOMICS VS AMPLICON SEQUENCING

A

METAGENOMICS: SHORT SEQUENCE FRAGMENTS FROM ‘ALL’ DNA
AMPLICON: MULTIPLE COPIES OF FRAGMENTS FROM 1 TARGET GENE

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12
Q

CLASSICAL GENE CLONING? (THE BIOTECH TOOLBOX)

A
  • PLASMIDS CAN BE PURIFIED FROM BACTERIA
  • PLASMIDS ARE CIRCULAR PIECES OF DNA WHICH CAN BE USED TO CARRY GENES OF INTEREST
  • PLASMIDS HAVE RESTRICTION SITES WHICH WILL BE CUT BY RESITRICTION ENZYMES INTO REPRODUCIBLE PATTERNS (PLASMIDS ARE CUT OPEN; THERE IS A BREAK IN THE CIRCLE)
  • THIS CUTTING CREATEAS ‘STICKY ENDS’ OF DNA
  • THE STICKY ENDS ATTACH TO EACH OTHER BY BASE PAIRING, FORMING WEAK HYDROGEN BONDS; GENES OF INTEREST GET INSERTED INTO SOME OF THE PLASMIDS, FORMING RECOMBINANT PLASMIDS. AND OTHERS CLOSE RIGHT BACK UP, REMAINING UNCHANGED)
  • DNA LIGASE MAKES THE BOND PERMINANT BY ATTACHING NUCLEOTIDES TOGETHER WITH POSPHODIESTER BONDS
  • THE PLASMIDS ARE MIXED WITH THE BACTERIA AND TAKEN UP BY SOME OF THE BACTERIA IN A PROCESS CALLED TRANSFORMATION
  • IT IS IDENTIFIED WHICH BACTERIA TOOK UP THE PLASMIDS WITH THE GENE OF INTEREST, AND THEY ARE ALLOWED TO REPRODUCE
    (COLORING, ANTIBIOTICS ETC ARE USED)
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13
Q

EXAMPLES OF ALTERNATIVE NICHE APPROACHES TO GENE CLONING (ALTERNATIVE TO THE CLASSIC CLONING)? WHAT DROVE THEIR CREATION?

A
  • TA CLONING
  • GOLDEN GATE
  • TOPO
  • GATEWAY CLONING
  • LIGATION-INDEPENDENT CLONING

DRIVEN IN LARGE PART BY EVER CHEAPER AND MORE COMPLEX DNA SYNTHESIS CAPABILITIES AND LIMITATIONS OF RESTRICTION ENZYMES (CAN ONLY CUT A VERY SPECIFIC DNA PATTERN, IF THAT PATTER DOESN’T APPAEAR IN OUR GENE OF INTEREST, DIFFERENT ENZYME NEEDS TO BE USED)
- THE LAST 15-20 YEARS: NEXT EVOLUTION OF CLONING TECHNIQUES, PROPELLED BY THE HUMAN GENOME PROJECT

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14
Q

MAJOR CURRENT TOOLS IN GENE CLONING/EDITING?

A

CRISPR Cas 9 AND GIBSON ASSEMBLY

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15
Q

CRISPR Cas 9

A
  • GENE EDITING TECHNIQUE
  • Cas9 IS A MICROBIAL PRODUCT
    STEPS:
  • DETERMINE A TARGET SEQUENCE OF DNA
  • SYNTHETISE A ‘GUIDE RNA’ WHICH WILL TARGET AND BIND TO THAT SPECIFIC SEQUENCE
  • ENZYME Cas9 RECOGNISES THE GUIDE RNA AND BINDS TO IT, AND THEN ACTS AS ‘MOLECULAR SCISSORS’ AND CREATES A DOUBLE STRANDED DNA BREAK
  • THE CUT IS REPAIRED INTRODUCING MUTATION (BY E.G. DESIGNING A HOMOLOGOUS PIECE OF DNA AND INSERTING IT WHERE THE BREAK IS AND ALLOWING DNA REPAIR MACHINERY TO FIX THE BREAK ACCORDING TO THE SPECIFIC ‘INSTRUCTIONS’ IN THE SEGMENT WE INSERTED)
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16
Q

GIBSON ASSEMBLY?

A
  • MOLECULAR CLONING TECHNIQUE
  • SIMPLE, EASY
    1) TAKE 2 FRAGMENTS OF DNA THAT YOU WANT TO JOIN TOGETHER
    2) ADD OVERLAP VIA PCR
    3) THIS IS THEN ADDED TO A SINGLE REACTION MIX (GIBSON ASSEMBLY MASTER MIX —> INCUBATE AT 50 DEGREES) CONTAINING ENZYMES
    4) EXONUCLEASE ENZYME WILL CHEW BACK THE 5’ ENDS OF THE DNAs (ARTIFICALLY CREATES COMPLEMENTARY OVERHANG)
    6) FRAGMENTS WILL ANNEAL WHEN THE TEMPERATURE IS LOWERED THROUGH REGULAR HYDROGEN BASE PAIRING
    7) BUT WHAT’S LEFT ARE GAPS IN THE BACKBONE BETWEEN 5’ AND 3’ ENDS
    8) DNA POLYMERASE CLOSES THE GAPS
    9) DNA LIGASE SEALES THE BACKBONES
  • LOW RATE OF MISTAKES
17
Q

THE WEIZMANN PROCESS?

A
  • ONE OF THE EARLIEST EXAMPLES OF MODERN MICROBIAL MICROTECHNOLOGY
  • DEVELOPED IN THE BEGINNING OF 20TH CENTURY, FULL INDUSTRIALISATION OCCURED AFTER WWII (ACETONE WAS A KEY INGREDIENT FOR THE PRODUCTION OF CORDITE; SIMILAR TO GUNPOWDER)
  • YIELDS ACETONE, BUTANOL AND ETHANOL IN 3:6:1 RATION
  • USES SUGAR AS A SOURCE TO FEED ANAEROBIC CLOSTRIDIUM ACETOBUTYLICUM
  • WAS EXTREMELY LARGE FERMENTATION PROCESS OF HUGE INDUSTRIAL, SOCIAL AND ISTORICAL IMPORTANCE (LARGEST PLANTS HAD 96 GERMENTERS AT 96,000 GALLONS EACH)
18
Q

NAME ‘BOTOX’ COMES FROM?

A

A TRADEMARK NAME FOR THE POTENT NEUROTOXIN BOTULINUM TOXIN (TYPICALLY A/B SEROTYPE)

19
Q

BOTULINUM TOXIN - NAME, SOURCE, PROBLEMS, PRODUCTION AND USE IN MEDICINE?

A
  • A NATURALLY ENCODED TOXIN BELONGING TO SEVERAL MEMBERS OF THE CLOSTRIDIUM GENUS, NOTABLY C. BOTULINUM
  • BOTULISM WAS A MAJOR PH HAZARD —> food poisoning caused by a bacterium growing on improperly sterilized tinned meats and other preserved food, e.g. sausages —> ‘BOTULUS’ IS LATIN FOR SAUSAGE
  • TOXIN ONE OF THE MOST POWERFUL KNOWN TO SCIENE
  • IN EXTREMELY SMALL DOSES USED IN: MIGRAINES, HYPERHIDROSIS (EXCESSIVE SWEATING), MOTOR NEURONE DISEASE ETC BECAUSE IT CAUSES PARALYSIS OF MUSCLES BY TARGETING THE NERVOUS SYSTEM
  • EXTENSIVE USE IN COSMETOLOGY
  • COMERCIALLY STILL PRODUCED IN C. BOTULINUM BECAUSE VERY VERY SMALL DOSES ARE NEEDED
20
Q

VALUE OF 1g OF BOTOX?

A

$1,500,000,000

21
Q

WHAT ARE BIOREACTORS?

A

Bioreactor is defined as a vessel that carries out a biological reaction and is used to culture aerobic cells for conducting cellular or enzymatic immobilization.

22
Q

WHAT ARE FIRST CHOICE SCALING SYSTEMS IN BIOTECHNOLOGY? (WHAT IS USED TO PRODUCE LARGE AMOUNTS OF A SUBSTANCE)?

A

MICROBES SUCH AS E. COLI

23
Q

LIMITATIONS OF USING MICROBES IN BIOTECHNOLOGY?

A
  • LACK OF POST TRANSLATIONAL MODIFICATIONS (PMTs) —> E.G. THEY CAN’T NATURALLY GLYCOSYLATE THEIR PROTEINS WHICH MOST EUKARYOTIC SYSTEMS WILL DO, AND THIS CAN BE A PROBLEM WITH THERAPEUTICS WHERE NOT HAVING THE RIGHT GLYCOSYLATION PATTERNS CAN LEAD TO ADVERSE IMMUNE RESPONSES, INSOLUBILITY, INACTIVITY
  • CANNOT READILY FORM DI-SULPHITE BRIDGES (SO MORE COMPLEX EUKARYOTIC PROTEINS CAN BE MORE DIFFICULT TO PRODUCE)
  • MAL FOLDED PROTEINS WILL FORM INCLUSION BODIES
24
Q

USE OF VIRUSES IN BIOTECHNOLOGY?

A
  • NO USE AS PRODUCTION VEHICLES BECAUSE THEY DON’T HAVE A CELLULAR INTERIOR
  • A LOT OF USE AS TRANSFECTION VEHICLES (Transfection: the introduction of nucleic acids into eukaryotic cells, or more specifically, into animal cells)
  • HEAVILY USED IN CELLULAR GENE THERAPY
  • PHAGE USED FOR MANY YEARS IN PHAGE DISPLAY FOR SCREENING PROTEIN MOIETIES/CREATING ANTIBODIES
24
Q

USE OF VIRUSES IN BIOTECHNOLOGY?

A
  • NO USE AS PRODUCTION VEHICLES BECAUSE THEY DON’T HAVE A CELLULAR INTERIOR
  • A LOT OF USE AS TRANSFECTION VEHICLES (Transfection: the introduction of nucleic acids into eukaryotic cells, or more specifically, into animal cells)
  • HEAVILY USED IN CELLULAR GENE THERAPY
  • PHAGE USED FOR MANY YEARS IN PHAGE DISPLAY FOR SCREENING PROTEIN MOIETIES/CREATING ANTIBODIES
25
Q

WHAT WAS THE FIRST EVER GENE THERAPY APPROVED?

A

GENDICINE, 2003, CHINA

26
Q

GENDICINE?

A
  • FIRST EVER GENE THERAPY APPROVED, CHINA, 2003
  • PARTICULARLY FOR HEAD AND NECK SQUAMOUS CELL CARCINOMA
  • WORKS VIA CARRYING AND OVEREXPRESSING THE TUMOR SUPRESSOR GENE p53 WHICH ENCOURAGES APOPTOSIS OF CANCER CELLS
  • USES REPLICATION-INCOMPETENT ADENOVIRUS VECTOR (ADENOVIRUS - ‘E. COLI OF GENE THERAPY)
  • CCA 30 000 PEOPLE RECEIVED THIS THERAPY SO FAR
27
Q

SYNTHETIC BIOLOGY?

A
  • ‘MICROBIAL BIOTECHNOLOGY 2.0’
  • AN ATTEMPT T PUSH NATURE BEYOND AND CREATE TRUL ‘NEW’ RESOURCES

Synthetic biology is a field of science that involves redesigning organisms for useful purposes by engineering them to have new abilities. Synthetic biology researchers and companies around the world are harnessing the power of nature to solve problems in medicine, manufacturing and agriculture.

28
Q

BIOTECHNOLOGY?

A

Biotechnology is a broad area of biology, involving the use of living systems and organisms to develop or make products.
Biotechnology is a broad term encompassing the application of biological components or processes to advance human purposes

29
Q

% OF HUMAN PROTEINS THAT HAVE BEEN ‘DRUGGED’ (HAVE MEDICATIONS THAT TARGET THEM)?

A

<20%, MOSTLY ON CELL SURFACE
- THE ‘UNDRUGGABLE CELL PARTS ‘ARE USUALLY WITHIN CELLS AND COULD HAVE GREAT THERAPEUTIC POTENTIAL, BUT DELIVERING MEDICATION BEYOND CELL SURFACE IS A CHALLENGE

INF (40 decks)

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