BIOSCI.

101 Laboratory Course

TUTOR: DEMONSTRATORS:
This Course has six labs:

1. Enzymology 2. Gene Expression 3 Genetics 3. 4. Evolution 5. Blood Glucose 6. Photosynthesis

Reminder Lab Organisation

Have you handed in your pre-lab assignment? Things to bring to every laboratory: Shoes (covered toes) Manual (having read the lab!) Pen, marker pens and pencil Ruler Calculator Laboratory L b t C Coat t If you miss a lab fill out an Absence from Laboratory form, attach a medical certificate, hand it to the Course Coordinator - Mandy Harper.

Your assignment Sheet!

Answer as you go & write in pen. Answers are almost always y somewhere in the text in lab &/or lecture guide OR in the presentation! PAY ATTENTION Write carefully and tidily. We cant mark what we cant read. Get your assignment sheet SIGNED OFF before you leave

Exercise 2.1 (pg 2.5): RNA hydrolysis

Start with this exercise

Boil RNA in acid conditions (Use t tube be with ith black screw cap) Acid hydrolysis breaks phosphodiester linkage produces mixture of nucleotides Separate nucleotides by chromatography

Laboratory is in two parts

1. Nucleic Acid structure 1 2. Control of Gene expression
Read each shaded box in the lab manual before trying to interpret each experiment

The Central Dogma of molecular biology

Basically once information is converted to protein, it cannot flow back to nucleic acid

DNA

In the cell information is stored where?

Transcription
mRNA ribosome

Translation
Copyright 2002 Pearson Education, Inc., publishing as Benjamin Cummings

protein polypeptide

Nucleic acids store and transmit hereditary information

There are two types of nucleic acids: ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). DNA provides direction for its own replication. DNA also directs RNA synthesis and, through RNA, controls protein synthesis.

A nucleic acid strand is a polymer of nucleotides

Each nucleotide consists of three parts

Base + Sugar = N l Nucleoside id

Another name for a nucleotide is nucleoside monophosphate

There are two types of nucleotides

6-membered ring

6-membered ring joined to a 5membered ring

RNA

phosphodiester bond

DNA

phosphodiester bond

DNA base pairing

Base pairing: A T G C

The sugars of one nucleotide are joined to the phosphate of the next nucleotide via This creates a sugar phosphate backbone

PHOSPHODIESTER BONDS

Base pairing: A T G C

Nucleic Acid Structure Important Terms Denaturation

De = Un, Nature = natural state or configuration changing changing something from its natural state state GENTLE BREAK WEAK BONDS i.e. HYDROGEN BONDS BETWEEN BASES

Hydrolysis From Greek: Hydro = water, Lysis = break breaking using water
HARSH WILL BREAK PHOSPHODIESTER LINKAGES

Hydrolysis
The covalent bonds connecting monomers in a polymer are disassembled by hydrolysis In hydrolysis as the covalent bond is broken a hydrogen atom and hydroxyl group from a split water molecule attaches where the covalent bond used to be

HYDROLYSIS DENATURATION

Inheritance is based on replication of the DNA double helix

DNA is relatively stable, hence ANCIENT DNA

DNA double stranded double helix RN single RNA l stranded d d extra OH H group (in ( presence of OH radicals) can attack the phosphodiester bond - Spontaneously at neutral pH and is called autocatalytic-cleavage

Compared to DNA, RNA is relatively unstable RNA single stranded & extra OH group relatively unstable OH group can attack the phosphodiester bond occurs spontaneously at neutral and pH (auto-catalytic-cleavage) ( y g ) alkaline p

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Precipitation and denaturation of DNA/RNA

RNA and DNA have different properties due to sizes and single/double-stranded single/double stranded nature. To investigate this: 2.2 Add ethanol and NaCl to DNA/RNA and observed any precipitate that forms 2.3 Place DNA and RNA in boiling water bath for ~5 minutes and repeat

Back to the RNA hydrolysis

The covalent bonds connecting monomers in a polymer are disassembled by hydrolysis In hydrolysis as the covalent bond is broken a hydrogen atom and hydroxyl group from a split water molecule attaches where the covalent bond used to be

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Chromatography and precipitations

Start precipitations exercises 2.2 and 2.3 Hydrolysis cool for 10 minutes while you wait load the standards on your plates then spot the plates with the Tube A samples ALL DONE? Pick up bacterial plates and start reading ahead, there will be a lac operon talk

2.1 RNA hydrolysis

phosphodiester bonds broken RESULT: ribonucleotides

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RNA hydrolysed to ribonucleotides

purines nucleotides unstable, breakdown to release bases

Four RNA hydrolysis products

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RNA hydrolysis (2.1)

After hydrolysis cool for 10 minutes while hil you wait it load l d th the standards t d d on your plates then spot the plates with the Tube A samples

Thin-layer chromatography

Load 4x standards

Load sample (4 (4-5x) 5x)

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RNA hydrolysis (2.1)

Afer the plates have dried, place in tanks (2 g ( groups/tank) p ) under supervision p Develop for as long as possible (> 1 hour), i.e. visualise ~4:30pm

Thin-Layer Chromatography standards

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RNA hydrolysis (2.1)

View in UV viewing box (bases absorb/fluoresce b b/fl under d UV light) li ht)

Thin-layer chromatography
solvent will carry different molecules at different rate

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Thin-layer chromatography

Chromatography tanks

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Viewing Chromatograms

Lab 2 Gene Expression The Lac Operon

2 2.4 4 Examine plates and determine genotype of stains A, B, C 2.5 Induce -galactosidase with lactose/IPTG & determine effect of glucose

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B Plate
Copy this slide and then make your own using this format.

B+I Plate
Copy this slide and then make your own using this format.

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lac Operon in E. coli

negative gene regulation
repressor gene

positive gene regulation via cAMP receptor protein

promoter operator p lacZ lacY lacA

mRNA cAMP Low glucose CAP lactose Transcription of -gal (inducer) occurs: Lactose (inducer) is present repressor Inactive repressor And glucose protein is absent + (cAMP is high) lactose

mRNA
permease transacetylase

-gal = enzyme

lactose galactose

glucose

lac Operon in E. coli

repressor gene promoter operator p lacZ mRNA
permease transacetylase

lacY

lacA

IPTG (inducer)

-gal = enzyme

repressor protein

Inactive repressor + IPTG

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lac Operon in E. coli

promoter operator p lacZ mRNA
permease transacetylase

lacY

lacA

-gal = enzyme

BCIG galactose

BCI

BLUE PRODUCT

lac Operon in E. coli

promoter operator p lacZ mRNA
permease transacetylase

lacY

lacA

-gal = enzyme

ONPG galactose

ONP

YELLOW PRODUCT

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2.4 Different strains of E.coli

Analysis of plates Think: in which plate is inducer present?
B+I plate, no inducer on B plate

Think: what do mutations do?

-galactosidase mutation:
non-functional enzyme

Operator mutation:
repressor cannot bind i.e. cant switch off

What colour will strains be?

Wild-type Wild t
white without inducer, blue with inducer

-galactosidase mutation
always white

Operator mutation
always blue

2.5 Induce -galactosidase

Add E. E coli (step 1) 1), followed by components to tubes as given in book (step 2) Incubate for 20 minutes at 37C, add lysis medium & tap Incubate for 15 minutes at 37C Add ONPG and incubate for 5 minutes Observe colour of each tube, note colour in some tubes may be pale Start ASAP, can do other tasks while waiting for incubations

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FINAL CHECKLIST:
Assignment g - name; stream number;
bench (#)

Clean Up instructions on
whiteboard.

Wash hands before leaving you have been handling bacteria

Place IN CORRECT CONTAINERS BY SINK

a) colour coded test tubes. b) ) black screw cap p tube ( (Kimble) ) c) boiling tubes (red tape around rim) d) blue pipette tips (Return empty beaker to your bench). e) colour coded pasteur pipettes.

Pl Place agar plates l t on t tray on side id b bench. h Stack white tray on side bench.

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XXXX MASTER SLIDE

Copy this slide and then make your own using this format.

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