Bacterial Genetics

Question 1

Prokaryote’s Genome Structure

Answer 1

No nucleus (no membtane bound organelles), plasmid (loop of DNA). Plasmid contains mainly non-repetitive, coding DNA. Either all genes are expressed or none of them are (single promoter). Proteins are produced in cell but can’t be easily removed (due to wall). No posttranslation modifications (folding, glycolysation, ect)

Question 2

Different Methods of Bacterial DNA transfer

Answer 2

Asexual reproduction (not mitosis), conjugation (DNA tranfered from 1 cell to another) and transformation (pieces of DNA taken up by membrane/wall)

Question 3

Gene expression in Eukaryotes

Answer 3

Genes are controlled individually. 2 types: constitutive (always expressed) and inducible (enviornmental expression). Requires multiple promoters to regulate genes

Question 4

Components of Eukaryotic Gene Expression

Answer 4

Promoters, Operators, Regulatory proteins, allosteric effectors

Question 5

Promoter Outline

Answer 5

RNA polymerase binding site on (supports transcription)

Question 7

Operator Outline

Answer 7

DNA sequence overlapping with/downstream of the promoter. Either a positive/negative gene regulator dependent on whether it’s an activator/repressor protein binding site.

Question 8

Regulatory Proteins Outline

Answer 8

Bind to DNA sequences either supporting or preventing transcription

Question 9

Eukaryote’s genome Structure

Answer 9

Nucleus. Contains mostly repetitive non-coding DNA. Genes are expressed independent of eachother. Proteins produced in cells are easily removed and have posttranslational modifications

Question 10

Allosteric Effectors Outline

Answer 10

Bind regulatory proteins at site other then binding. Changes confirmation so that, it can no longer bind to operator

Question 11

Activators and positive operators

Answer 11

If an activator binds to positive operator, transcription is supported (required for transcription). If there is no binding transcription is repressed

Question 12

Repressors and negative operators

Answer 12

If repressor binds transcription is repressed. If no binding transcription is supported (required for transcription)

Question 13

Lac Operon Outline

Answer 13

Encodes genes for Lactose metabolism. Contains 3 inducible genes (A,Y,Z) all which must be transcribed to mRNA (all same mRNA) for lactose metabolism and 1 constitutive (I) that acts as negative feedback

Question 14

Lac Gene Y Outline

Answer 14

Produces permease. Transports lactose to cell

Question 15

Lac gene X Outline

Answer 15

Produces Beta Galactosidase. Cleaves lactose into gucose + galactose

Question 16

Lac gene A Outline

Answer 16

Produces transacetylase

Question 17

Lac gene I Outline

Answer 17

Produces Lac represor. Binds to operating site preventing A,Y and Z expression

Question 18

What is the allosteric inhibitor for Lac Repressor Protein

Answer 18

Lactose. Binds to induce confirmational change. Allows expression of genes for it’s own metabolism

Question 19

Sticky Restriction Enzymes Examples

Answer 19

EcoR1 and BamH1. Makes for easier editing (complimentary overhang)

Question 20

Blunt Restriction Enzymes Example

Answer 20

Sma1. Cuts at same location (difficult editing as no exposed nucleotide bases)

Question 21

Competent Cells Def

Answer 21

Cells capable of taking up novel DNA-plasmid vector

Question 22

Alpha Complementation Def

Answer 22

Combining 2 parts of a protein (ie specific DNA sequence + plasmid for complete gene set) to make complete functional proteins

Question 23

Alpha Complementation in E Coli

Answer 23

If cells contain Lac gene Z deletion it can’t produce functional active beta-galactosidase. Can fuse plasmid with functional lac Z gene for uptake by cells to (when successful) produce active beta galactosidase. Contains antibiotic ressistant gene, so survive in antibiotic conditions while non-competent cells don’t

Question 24

Blue/White Screening Outline

Answer 24

Way to identify competent (cells that sucessfully take up recombinnat protein) in lac operon, to measure lac operon disruption

Question 25

Blue Colonies Outline

Answer 25

Active beta galactosidase has been formed -> Lac operon not disrupted by new DNA (failed DNA uptake/ DNA uptake to plasmid region other then operon)

Question 26

Blue/White Screening Method

Answer 26

Isopropyl-beta-D-1 thiogalactopyranoside acts as activator in lac operon. Once genes are activated (and beta galactosidase should have been formed) x-gal is administered as substrate. If x-gal is cleaved by active beta galactosidase then a blue colour is released

Question 27

White Colonies Outline

Answer 27

non-active beta galctosidase formed -> lac operon disrupted by DNA insertion (DNA uptake in operon)

Question 28

Expression Vectors Outline

Answer 28

Produce a protein from a cloned gene

Question 29

Expression Vector Considerations in all celll types

Answer 29

promoters/enhancers present, correct sequence length (prevent condon framing shift), protein dependent (need to know type)

Question 30

Expression Vector Consideration in Eukaryotic Cells

Answer 30

correct protein folding, post translation modifications, difficulty in introducing foreign DNA (no transformation)

Question 31

Viral Vector Considerations

Answer 31

Higher transfection efficiency in mammalian cells then bacterial plasmids. require higher safety measures (highly pathogenic) and thus more expensive