Cloning DNA into Plasmid Cloning DNA into Plasmid Vectors and Sequencing.Vectors and Sequencing.

ABE Workshop 2007ABE Workshop 2007

Josh NelsonJosh Nelson

26 – Jun – 200726 – Jun – 2007

What is Cloning?What is Cloning?

Making multiple identical copies of a geneMaking multiple identical copies of a gene This can be placed into a vectorThis can be placed into a vector This in turn can be placed into a host This in turn can be placed into a host

bacteriabacteria Modern plasmid vectors are designed to Modern plasmid vectors are designed to

make the process fast and easymake the process fast and easy Inclusions of antibiotics and other Inclusions of antibiotics and other

screening methods ensure correct gene screening methods ensure correct gene cloningcloning

Steps in the Cloning Process Steps in the Cloning Process

1.1. Isolate ProteinIsolate ProteinChoose a vector

Isolation of ProteinIsolation of Protein

Amino acid sequencingAmino acid sequencing

Purify proteinPurify protein

Sequence proteinSequence protein

Design primerDesign primer

Run PCRRun PCR

Result: DNA of interestResult: DNA of interest

Isolate Protein PDI

Start with PDIStart with PDI

Obtain sequence from Obtain sequence from databasedatabase

Make primersMake primers

Run PCRRun PCR

Steps in the Cloning Process Steps in the Cloning Process

1.1. Isolate ProteinIsolate Protein

2.2. Choose and Choose and Prepare VectorPrepare Vector

Choosing and Preparing the VectorChoosing and Preparing the Vector

pCR 4 – TOPO pCR 4 – TOPO • Small plasmidSmall plasmid• LacZ – ccdBLacZ – ccdB

Selective for ccdB geneSelective for ccdB gene

Insert PCR ProductInsert PCR Product• A overhangA overhang• Vector has T overhangVector has T overhang

LigationLigation• TopoisomeraseTopoisomerase

Steps in the Cloning Process Steps in the Cloning Process

1.1. Isolate ProteinIsolate Protein

2.2. Choose and Choose and Prepare VectorPrepare Vector

3.3. Transform intoTransform into HostHost

Transforming into Expression Host Transforming into Expression Host

E. Coli CellsE. Coli Cells• Soak in CaClSoak in CaCl22

Make them Make them competentcompetent

Insert vectorInsert vector• Heat shockHeat shock

Spread on culture Spread on culture plateplate• Kanamycin added Kanamycin added

for selection for selection Incubate Incubate

Steps in the Cloning Process Steps in the Cloning Process

1.1. Isolate ProteinIsolate Protein

2.2. Choose and Choose and Prepare VectorPrepare Vector

3.3. Transform into Transform into HostHost

4.4. Extraction of Extraction of Cloned GeneCloned Gene

Extraction of Cloned GeneExtraction of Cloned Gene Choose coloniesChoose colonies

• WhiteWhite

Place in LB mediumPlace in LB medium• Contains Ampicillin & Contains Ampicillin &

KanamycinKanamycin• IncubateIncubate

Isolate cloned geneIsolate cloned gene• Cell pelletCell pellet• Gene pelletGene pellet

Run gel to confirm you Run gel to confirm you cloned correct genecloned correct gene

Blue/white?White

Blue

Steps in Cloning the Process Steps in Cloning the Process

1.1. Isolate ProteinIsolate Protein

2.2. Choose and Choose and Prepare VectorPrepare Vector

3.3. Transform into Transform into HostHost

4.4. Extraction of Extraction of Cloned GeneCloned Gene

5.5. SequenceSequence

SequencingSequencing

Add primers to DNAAdd primers to DNA• T7 upstreamT7 upstream• T3 downstreamT3 downstream

DideoxynucleotidesDideoxynucleotides• Terminates synthesis Terminates synthesis

Fluorescent DyesFluorescent Dyes• Help in fast sequencingHelp in fast sequencing• Dyes different weightsDyes different weights

Polyacrylamide Gel Polyacrylamide Gel ElectrophoresisElectrophoresis

Results of SequencingResults of Sequencing

One was PDI 1One was PDI 1• White colonyWhite colony

One was vectorOne was vector• Base pairs between Base pairs between

1550 and 23001550 and 2300• ccdB gene not ccdB gene not

expressedexpressed

My ApplicationMy Application

Shrimp aquacultureShrimp aquaculture

Microbial communityMicrobial community

Build up of ammoniaBuild up of ammonia

Clone gene which expresses desired Clone gene which expresses desired trait trait