IM_Midterm

Question 1

What are “Industrial “ Microorganisms

Answer 1

Yeast
Bacteria
Fungi
Mammalian cell

Question 2

Characteristics of Ideal Industrial microorganism

Answer 2

*Can be grown easily in the lab
* Can grow on and utilize cheap substrates
* Can produce a diversity of products
* Can produce the desired product in less time
* Amenable for genetic manipulation
* GRAS (generally regarded as safe); non-pathogenic

Question 3

Why clone genes?

Answer 3

Make Proteins
Make mutants

Question 4

Where do Taq polymerase originate

Answer 4

Thermus aquaticus

Question 5

3 steps in PCR

Answer 5

Denaturation, Annealing, Extending stage

Question 6

Components of PCR mastermix

Answer 6

DNA polymerase, dNTPs, PCR buffer, MgCl2, mol grade water.

Question 7

If u have PCR mastermix, what are the remaining components to proceed with PCR?

Answer 7

Template DNA, Forward primer, Reverse primer

Question 8

Full length of 16srRNA

Answer 8

1500 bp (1504 bp)

Question 9

A reliable molecular marker for
identification of bacteria and archaea, which is highly conserved.

Answer 9

16srRNA

Question 10

Molecular cloning workflow steps

Answer 10

Vector Preparation, Insert preparation, Ligation, Transformation, Colony screening

Question 11

Used for verification of PCR products

Answer 11

Agarose gel electrophoresis

Question 12

Small circular DNA with <5-10 kb insert range

Answer 12

Plasmid

Question 13

Linear viral DNA with up to 20 kb insert range

Answer 13

Bacteriophage

Question 14

Hybrid of plasmid and phage with up to 50 kb

Answer 14

Cosmid

Question 15

DNA containing yeast centromere, telomeres, and origin of replication with 200 up to 1000 kb insert range

Answer 15

Yeast artificial Chromosome (YAC)

Question 16

Types of plasmids

Answer 16

Fertility plasmid, Col plasmid, Resistance plasmid, Degradative plasmid, Virulence plasmid

Question 17

Why use plasmid for molecular cloning?

Answer 17

1. Ability to replicate into host cells
2. Unique restriction enzyme sites for insertional
   cloning
3. Genetic marker to select for host cells
   containing the vectors.
4. Low molecular weight

Question 18

Type of plasmid (copy number) which is replicated frequently, and is used for cloning and protein expression

Answer 18

High copy number

Question 19

Type of plasmid (copy number) which is replicated slowly, and is used for expression of toxic products, and mutant studies

Answer 19

Low copy number

Question 20

3 main parts/regions of plasmid

Answer 20

Origin of replication (ORI), Multiple cloning site, Antibiotic resistance gene

Question 21

A low copy number plasmid with 2 antibiotic resistance genes (ampR, tetR) derived from naturally occuring vectors (R1,R-65 respectively), and ORI from pMB81

Answer 21

pBR322

Question 22

promoter’s regulatory action which signal to turn ON synthesis of appropriate
enzyme

Answer 22

Inducible

Question 23

promoter’s regulatory action which signal to turn OFF synthesis of appropriate
enzyme

Answer 23

Repressible

Question 24

synthesis of enzymes is “on” at all times.

Answer 24

Constitutive

Question 25

Differentiate strong vs weak promoter

Answer 25

Weak promoter: little (or infrequent) RNA polymerase binding → low levels of transcription → low levels of corresponding protein. Strong Promoter: lots of (or frequent) RNA polymerase binding → high levels of transcription → high levels of corresponding protein.

Question 26

Short sequences in MCS which may be cut with these enzymes

Answer 26

Restriction enzymes

Question 27

Differentiate single digestion and double digestion

Answer 27

Single digestion uses one type of restriction enzyme to "cut" a fragment of plasmid, while Double digestion uses 2 different RE.

Question 28

In blue white screening, which colonies should you pick to know whether the insert is successfully ligated in your plasmid

Answer 28

White colonies

Question 29

Which gene is responsible for blue-white screening?

Answer 29

lacZ gene

Question 30

During ligation process, which step should you do to avoid plasmid religation or recircularization?

Answer 30

Dephosphorylation

Question 31

What is the advantage of Extracellular protein product over intracellular ?

Answer 31

Easier to collect, product is on the supernatant, Does not require sonication

Question 32

What is the unit ng protein eme

Answer 32

Dalton

Question 33

2 ways to make a competent cell

Answer 33

Electroporation, Chemical competency

Question 34

Ligation reaction composition

Answer 34

Vector, and Insert + Ligase reaction buffer, dH2O, Ligase

Question 35

Optimal ratio of insert to vector

Answer 35

3:1 to 5:1

Question 36

Removes 5'- and 3' - phosphates from DNA, RNA, and dNTPs

Answer 36

Calf Intestinal alkaline phosphatase

Question 37

Protein product of lacZ gene

Answer 37

Beta-D-Galactosidase

Question 38

Screening plate for blue-white screening composition

Answer 38

LB agar + Ampicillin (any antibiotic present in the vector plasmid) + X-gal

Question 39

structurally mimics lactose and is used to induce protein expression

Answer 39

Isopropyl β-D-1-thiogalactopyranoside (IPTG)

Question 40

Protein detection through Separation of proteins proteins according to their molecular weight (Similar to AGE)

Answer 40

Sodium Dodecyl Sulfate– Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Question 41

a laboratory method used to detect specific protein molecules from among a mixture of proteins

Answer 41

Western blot

Question 42

Sources of microorganism through culture collection

Answer 42

American Type Culture Collection (ATCC) German Collection of Microorganisms and Cell Cultures (DSMZ) Japan Collection of Microorganisms (JCM) Philippine National Collection of microorganisms (PNCM-BIOTECH, UPLB)

Question 43

Sources of microorganisms for industrial use

Answer 43

Terrestrial, Aquatic/Marine, Culture collection

Question 44

isolation and screening methods

Answer 44

Sample Collection and Processing Serial Dilution and Plating Purification Preservation Screening Assays

Question 45

Screening for Antimicrobial Activity: Test Strains for gram negative, gram positive, yeast, fungi

Answer 45

Gram Negative Representative (e.g. Escherichia coli) Gram Positive Representative (e.g. Staphylococcus aureus) Yeast Representative (Candida albicans) Fungi Representative (Aspergillus niger)

Question 46

Point of which growth of microorganism is inhibited

Answer 46

Minimum inhibitory concentration (MIC)

Question 47

lowest concentration that kills the bacteria i.e. no colony growth

Answer 47

Minimum Bactericidal Concentration (MBC)