Application of molecular technology in biotechnology

molecular cloning technique and its application in food biotechnology.

1. Molecular biology application in food biotechnology Part 2. Brief story of Molecular Cloning and its application Pramono , H.

2. Outline Molecular cloning DNA extraction Electrophoresis PCR Cloning Gene expression Example

3. DNA extraction DNA isolation consist of whole genome and gene isolation Whole genome isolation is first step to isolate gene from organism/cells DNA which are negative charged always wrapped by cell membrane and some times other partition (cell wall/ peptidoglycan ) Principles: disruption of cell membrane and wall separate the genome from other substance Isolate genomic DNA

4. Basic Protocol Most DNA extraction protocols consist of two parts A technique to lyse the cells gently and solubilize the DNA Enzymatic or chemical methods to remove contaminating proteins, RNA, or macromolecules In plants, the nucleus is protected within a nuclear membrane which is surrounded by a cell membrane and a cell wall. Four steps are used to remove and purify the DNA from the rest of the cell. Lysis Precipitation Wash Resuspension

5. A comparison of DNA extraction methods used in research labs as opposed to classroom labs Research Lysis : grind in Liquid N 2 and use detergent Precipitation Part I : phenol/chloroform extraction to get rid of proteins Precipitation Part II : addition of salts to interrupt hydrogen bonding between water and phosphates on the DNA Precipitation Part III : addition of ethanol to pull DNA out of solution Wash and resuspend : DNA is washed in ethanol, dried, and resuspended in H20 or TE buffer. Classroom Lysis : grind in mortar/ pestel and use detergent Precipitation Part I : NONE (chemical are too dangerous!) Precipitation Part II : addition of salts to interrupt hydrogen bonding between water and phosphates on the DNA Precipitation Part III : addition of ethanol to pull DNA out of solution Wash and resuspend : DNA is washed in ethanol, dried, and resuspended in H20 or TE buffer.

6. Break down the cell wall and membranes Centrifuge to separate the solids from the dissolved DNA Precipitate the DNA using isopropanol Centrifuge to separate the DNA from the dissolved salts and sugars Wash the DNA pellet with Ethanol and dry the pellet Dissolve DNA Overview of DNA Extraction

7. 1 kbp and 100 bp ladders Genomic DNA of 5 species of cereals Expected Results in a Research Lab Below is an agarose gel that has 5 genomic DNA samples from various plants. Note that the DNA runs at a very high molecular weight and as a clear, thick band. This DNA was extracted in a research lab under optimal conditions

8. Gel Electrophoresis Electrophoresis is method to separate substance (commonly DNA or Protein) employing agarose or PAGE Negative charged molecule will move to positive part of electrophoresis chamber Usually adding Etidium bromide ( EtBr ) or another substance that interfere in DNA for visualization purposes

9. waynesword.palomar.edu

10. UV light visualization enfo.agt.bme.hu

11. PCR Polymerase chain reaction (PCR) is a method to replicate DNA Polymerase (remember replication) from Thermus aquaticus ( Taq polymerase) commonly uses to elongate process since this enzyme still firm in ±95 o C Invented by Karri Mullis

12. www.slideshare.net

13. Principles of PCR www.soi.wide.ad.jp

14. Molecular cloning Cut the gene Paste the gene on vectors Transform into E.coli Screening process Purification

15. Gene Cloning molecularhub.blogspot.com

16. Cloning Vector Cloning vector can be: Bacteriophage Plasmid Cosmid Other

17. Bacteriophage

18. Cosmid

19. Plasmid

20. Lodge et al.

21. Expression Little bit different with cloning Using different vector, called expression vector Expression vector can be: E. coli S. cereviceae B. subtilis Need induction

22. www.biomics.cn

23. Review https://www.youtube.com/watch?v=sjwNtQYLKeU

24. Application of technology Detection of pathogenic bacteria in food sample Isolating important gene and express its protein in mass production Bioreactor of some specific and useful protein Screening of bioactive-producing isolates

25. Cloning

26. Detection

27. Protein Profiling

28. Home work (group of 5) Please make short review (max 12 p) included: Basic molecular biology of the cell DNA extraction, PCR, gene Cloning, cloning vectors, gene expression, gel electrophoresis, PAGE Application of molecular technique in food/fishery technology (minimum explain 5 journal from Sciencedirect , springerlink , or DOAJ) Rule: A4, Arial 11, margin 4333, Consist of Introduction, summary of theory, application of theory (5 journal)

29. Any question?