Chapter 2A How do you begin to clone a gene? - PowerPoint Presentation

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Uploaded On 2022-05-14

Chapter 2A How do you begin to clone a gene? - PPT Presentation

Tab C abridged sequence Starts on page C1 Student Introduction Reading Plasmids and Restriction Enzymes Activity Clone that gene Laboratory 2a Preparing to verify the rfp gene digesting the ID: 911041

gene plasmid restriction dna plasmid gene dna restriction enzymes sticky promoter para antibiotic rfp recombinant genes clone cut sequence

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Slide1

Chapter

How do you begin to clone a gene?

Slide2

Tab C- abridged sequence

Starts on page C-1StudentIntroductionReading:

Plasmids and Restriction Enzymes

Activity: Clone that geneLaboratory 2a: Preparing to verify the rfp gene: digesting the pARA-R plasmidQuestionsGlossary

Student guides

Slide3

Tab

C: abridged sequenceStarts on page C-1

Teacher guide

Slide4

Already mastered the micropipette and gel electrophoresis

Knowledge of the beginning of ABE curriculumInsulin, diabetes, etc.Structure of DNA

Nucleotide structure in detail

Base pairing and hydrogen bondsDecoding DNA:TranscriptionTranslation Previous knowledge and skills

Slide5

Describe the characteristics of plasmids

Explain how plasmids are used in cloning a geneDescribe the function of restriction enzymesExplain how to use restriction enzymes to create a recombinant

plasmid

Chapter 2A goals

Slide6

Session 1 – 45 minutes

Review chapter goalsAnswer the What do you already know

questions

Have students read Plasmids and Restriction Enzymes and answer the CONSIDER questionsLead a discussion on student answersSession 2 – 45 minutesStudents complete Clone that gene exerciseAnswer the Stop and Think questionsSession 3 – 45 minutes

Students complete Laboratory 2a

Answer all of the corresponding

questions

Proposed sequence of activities

Slide7

QUESTION: Why is circular better

?!?!

Bacteria CAN have 2 forms of DNA:

ChromosomeSingle loop of DNACondensed and segregated into a region called nucleoidContains all information necessary for lifePlasmid Small loop of DNANonessentialExtrachromosomal – “outside” the chromosome

Vectors

Replicate autonomously

Bacterial DNA

Slide8

Power of plasmid as vector

recognized… but how to incorporate foreign gene of interest??

Transformation

Uptake of DNA from environmentConjugationTransfer of DNA from one bacterium to anotherTransduction Transfer of DNA via a bacteriophageExogenous genetic transfer of bacteria

http://

www.hhmi.org/biointeractive/bacterial-conjugation

Slide9

Plasmid Vector

Ori

Sequence where enzymes bind to

Initiate DNA replicationAbility to undergo transcriptionPromoter sequence binds RNA polymeraseAll genes have

promoters

next them (must be together)

Antibiotic resistance

Genes for antibiotic resistance

common

Genes code for proteins that inhibit action of

antibiotics

Secreted by microbes against microbes

Make it a useful tool in identifying bacteria

Restriction

enzymes

Plasmid as the ideal vector for genetic engineering

Slide10

Bacteriophage

Some E. coli resistant to infection by a bacteriophage

Let to discovery of restriction enzymes

Proteins that restrict growth of bacteriophageRecognize and destroy phage DNA without damaging host cellRecognition sitesSpecific sites DNA is cut (digestion) at

Restriction Enzymes

Slide11

Restriction Enzymes

Must cut at correct site to ensure no genes are disturbed

Keep

Ori, promoter, and gene for antibiotic resistanceMust cut plasmid near promoter so inserted gene expressedCut human DNA close to gene of interestGene for insulinCut asymmetrically to create sticky endsSingle stranded overlapping endImportant when reannealing sequences

Recognition sites are palindromes

What's in a name??

Slide12

Blunt Ends

“Sticky” Ends

STICKY ENDS

Slide13

pALINDROMES

Palindromes are the same forwards/backward:

A nut for a

of tuna

Eva, can I

sta

ats

in a

cave

Slide14

Palindromes result in sticky

ends - form hydrogen bonds

easily

Slide15

sticky end

Bam

H I

sticky end

Hin

d III

sticky end

Bam

H I

sticky end

Hin

d III

ENGINEERING THE PLASMID

LIGATION OF RFP GENE

Slide16

Plasmid + restriction enzymes = Recombinant Plasmid

Contains portions of original plasmid and “new” DNA sequence

Insert desired gene

DNA ligaseChimera GMO!!

Slide17

Activity - Clone that Gene

Make a paper model of a recombinant plasmid that contains insulin gene

Three tasks:

Cut the plasmid and the human DNA with correct restriction enzymes

Insert insulin gene into plasmid

Determine which antibiotic you would use to ID bacteria

that have taken in the plasmid

Slide18

Slide19

Alternatives

? Pipe cleaner activity

Slide20

pARA

-R plasmidrfp geneRed fluorescent protein

Use this as our insulin

modelHow does this all apply to ABE?

Slide21

Chapter 2A

How do you begin to clone a gene?

Slide22

Restriction digest

Use restriction enzymes to cut plasmid

Ensure recombinant plasmid is correct one for making red fluorescent protein

pARA-RLength of fragments can be determined by gel electrophoresisPurpose:

Slide23

ampR

Ampicillin resistance

Red fluorescent protein (

rfp) genePromoterInitiate transcriptionOri siteInitiate DNA replicationaraCSequence that activates the promoter when arabinose present in environmentpBAD

Regulatory molecule for the arabinose

promoter

pARA

-R

plasmid = Recombinant

plasmid

Slide24

pARA-R plasmid – how does it work?

Activator (

araC

) controls promoterArabinose present:Sugar binds to promoter and changes shape

Promoter binds RNA polymerase = transcription