1. Cloning DNA into Plasmid Vectors and Sequencing. ABE Workshop 2007 Josh Nelson 26 – Jun – 2007

2. What is Cloning? Making multiple identical copies of a gene Making multiple identical copies of a gene This can be placed into a vector This can be placed into a vector This in turn can be placed into a host bacteria This in turn can be placed into a host bacteria Modern plasmid vectors are designed to make the process fast and easy Modern plasmid vectors are designed to make the process fast and easy Inclusions of antibiotics and other screening methods ensure correct gene cloning Inclusions of antibiotics and other screening methods ensure correct gene cloning

3. Steps in the Cloning Process 1. Isolate Protein Choose a vector

4. Isolation of Protein Amino acid sequencing Amino acid sequencing Purify protein Purify protein Sequence protein Sequence protein Design primer Design primer Run PCR Run PCR Result: DNA of interest Result: DNA of interest Isolate Protein PDI  Start with PDI  Obtain sequence from database  Make primers  Run PCR

5. Steps in the Cloning Process 1. Isolate Protein 2. Choose and Prepare Vector

6. Choosing and Preparing the Vector pCR 4 – TOPO pCR 4 – TOPO Small plasmidSmall plasmid LacZ – ccdBLacZ – ccdB Selective for ccdB gene Selective for ccdB gene Insert PCR Product Insert PCR Product A overhangA overhang Vector has T overhangVector has T overhang Ligation Ligation TopoisomeraseTopoisomerase

7. Steps in the Cloning Process 1. Isolate Protein 2. Choose and Prepare Vector 3. Transform into Host

8. Transforming into Expression Host E. Coli Cells E. Coli Cells Soak in CaCl 2Soak in CaCl 2 Make them competent Make them competent Insert vector Insert vector Heat shockHeat shock Spread on culture plate Spread on culture plate Kanamycin added for selectionKanamycin added for selection Incubate Incubate

9. Steps in the Cloning Process 1. Isolate Protein 2. Choose and Prepare Vector 3. Transform into Host 4. Extraction of Cloned Gene

10. Extraction of Cloned Gene Choose colonies Choose colonies WhiteWhite Place in LB medium Place in LB medium Contains Ampicillin & KanamycinContains Ampicillin & Kanamycin IncubateIncubate Isolate cloned gene Isolate cloned gene Cell pelletCell pellet Gene pelletGene pellet Run gel to confirm you cloned correct gene Run gel to confirm you cloned correct gene Blue/white? White Blue

11. Steps in Cloning the Process 1. Isolate Protein 2. Choose and Prepare Vector 3. Transform into Host 4. Extraction of Cloned Gene 5. Sequence

12. Sequencing Add primers to DNA Add primers to DNA T7 upstreamT7 upstream T3 downstreamT3 downstream Dideoxynucleotides Dideoxynucleotides Terminates synthesisTerminates synthesis Fluorescent Dyes Fluorescent Dyes Help in fast sequencingHelp in fast sequencing Dyes different weightsDyes different weights Polyacrylamide Gel Electrophoresis Polyacrylamide Gel Electrophoresis

13. Results of Sequencing One was PDI 1 One was PDI 1 White colonyWhite colony One was vector One was vector Base pairs between 1550 and 2300Base pairs between 1550 and 2300 ccdB gene not expressedccdB gene not expressed

14. My Application Shrimp aquaculture Shrimp aquaculture Microbial community Microbial community Build up of ammonia Build up of ammonia Clone gene which expresses desired trait Clone gene which expresses desired trait