Estimation of BT Gene and BT protein by ELISA and PCR Screening

Slide 1- title
Slide 2- content
Slide 3- introduction about cotton plant
Slide 4- cotton plant types
Slide 5 - bacillus thurangenesis
Slide 6 - Needs of BT cotton
Slide 7 - Advantages and disadvantage of BT
Slide 8 - How BT protein is tested -ELISA
Slide 9 - ELISA reader
Slide 10 - Isolation of DNA from leaf, root, seed.
Slide 11- PCR Screening
Slide 12- Agarose gel electrophoresis
Slide 13 - UV gel documentation
Slide 14 - Tissue culture
Slide 15 - Thanks

1. TULASI SEEDS SHORT TERM INTERNSHIP ESTIMATION OF BT GENE AND BT PROTEIN BY ELISA AND PCR SCREENING   Submitted By : Kodiganti Pavithra
Group: II B.Sc Food Technology
Roll No: 21411009
Mentor : B. Dorka Vijaya Kumari   1

2. Introduction Cotton is a soft, fluffy staple fiber that grows in a boll, or protective case. Cotton cultivation is a significant agricultural industry in many countries, with the united states, China, and India being some of the largest cotton producers. It's a member of the malvaceous family and is primarily grown for its cotton bolls, which contain the valuable fibers There are different varieties of cotton, each with its unique characteristics and adaptability to specific environmental conditions. The cotton plant plays a crucial role in the global textile and agricultural industries, making it a subject of substantial economic and cultural importance.

3. Cotton Types Gossypium hirsutum : upland cotton, native to Central America, Mexico, the Caribbean Gossypium barbadense : known as extra-long staple cotton, native to tropical South America Gossypium arboretum : tree cotton, native to India and Pakistan Gossypium herbaceous : Levant cotton, native to southern Africa and the Arabian Peninsula

4. BACILLUS THURANGENESIS BT: A global biopesticide used via aerial sprays and transgenic plants BT: Spore-forming, Gram-positive soil bacterium BT Protein: Effective against 3 key insects Cry1Ac: American bollworm, pink bollworm, and lepidoptera species Cry2Ab: American bollworm, pink bollworm, and others Discovered by Monsanto A Sustainable Solution for Pest Control

5. NEEDS OF BT The insertion of the genes from Bt thuringiensis causes cotton plant cells to produce crystal insecticidal proteins, often referred to as Cry-proteins These insecticidal proteins are effective in killing some of the most injurious caterpillar pests of cotton, such as the larvae of tobacco budworms and bollworms Cotton varieties containing the Cry1Ac Bt proteinsn provide protection against three major U.S.cotton pests—tobacco bud- worms, bollworms, and pink bollworms.

6. Advantages Disadvantages Increased cotton yield by controlling American, Spotted, and Pink bollworms. Effective protection due to Bt gene's protein against bollworms. Reduced need for insecticide in Bt cotton cultivation. Higher cost compared to non-GMO crops. Potential disruption of natural gene flow. Risk of pest resistance to Bt toxins and declining crop production.

7. HOW BT PROTEIN IS TESTED ELISA, or Enzyme-Linked Immunosorbent Assay, is a widely used method to detect Bt protein in cotton plants. It involves an antigen-antibody reaction using enzymes.  In a 96-well plate, 93 wells are filled with plant samples, and 3 wells serve as controls. Monoclonal antibodies are present in the plate . During incubation, Bt protein binds to these antibodies. After washing, TMB substrate is added, leading to a blue color when peroxidase reacts with it, indicating a positive Bt result.  The addition of 1N HCl (stop solution) turns the color yellow due to a pH change, still indicating a positive Bt result.

8. ELISA READER ELISA reader used for Qualitative analysis of Bt protein OD at 405 and 450nm. OD values that indicates Bt protein positive are 0.2nm to 4nm OD values that indicates Bt protein negative are below 0.2nm

9. ISOLATION OF DNA DNA isolation is a technique to obtain pure DNA from samples, commonly used for Bt-positive plants. In this process, negatively charged DNA sticks to positively charged resin beads. Key tools are a water bath, centrifuge, refrigerator, and hot air oven. It employs columns and specific buffers: PL (Plant Lysis), PC (DNA Binding), PW (Plant Wash), and PE (Plant Elution). There are two types of columns, violet and green. Elution buffer is used to release DNA through centrifugation . After the process, you get 100µl of pure DNA, suitable for tasks like gel electrophoresis or PCR. S.NO CHEMICAL NAME REQUIRED VOLUME 1 Plant lysis buffer 1 (PL-1)_ 400 ul 2 Plant lysis buffer 2 (PL-2)_ 300 ul 3 RNase –A 10 ul 4 Plant lysis buffer 3 (PL-3)_ 75 ul 5 DNA binding buffer (PC) 450 ul 6 Plant wash buffer 1 (PW-1) 400 ul 7 Plant wash buffer 2 (PW-2) 700 ul 8 Plant Elution buffer 100 ul

10. POLYMERASE CHAIN REACTION (PCR) SCREENING PCR was developed by Kary Mullis in 1983. It’s a technique that exponentially amplifies a specific DNA region in vitro. PCR involves three key steps:
1. Denaturation: DNA is heated to 90-94°C for 10-15 seconds.
2. Annealing: Temperature is lowered to 50-60°C for 30 seconds to 2 minutes, allowing primers to bind to the DNA.
3. Extension: Temperature is set at 70-74°C for 2-3 minutes, DNA polymerase extends the DNA strands.
This process rapidly produces many copies of the target DNA sequence. S. No Chemical Name Required volume Cry1Ac 1 Master mix 7.5ul 2 Primer-1 0.3ul 3 Primer-2 0.3ul 4 Primer-3 0.3ul 5 Double distilled water 4.6ul 6 Isolated DNA 2ul Cry2Ab 7.5ul 0.15ul 0.15ul 0.3ul 4.9ul 2ul

11. AGAROSE GEL ELECTROPHORESIS Gel electrophoresis separates DNA fragments by size in a solid support medium such as an agarose gel. There is difference in the electrophoretic mobility of these charged molecules due to their difference in size ,shape and charge. Prepare 0.8% Agarose gel and add 13ul of Ethidium bromide , mix well pour in a tray . Place the tray in buffer tank , remove the comb and mix the isolated DNA with loading buffer . Load the sample in wells, run for 32 mins .After observe the bands in Gel documentation under UV light

12. Uv gel documentation helps to observe the DNA bands under UV light And zygosity confirmation of Bt gene by using formation of DNA bands Type of gene Base pairs Zygosity conformation Bt positive/negative Cry1Ac 800bp Homozygous Bt positive Cry2Ab 1800bp Homozygous Bt positive Cry1Ac 800bp + 1000bp Heterozygous Bt positive Cry2Ab 1800bp + 1000bp Heterozygous Bt positive UV GEL DOCUMENTATION

13. Tissue Selection: Choose plant tissue to be cultured, which can be a single cell, cell population, or part of an organ. Nutrient Medium Preparation: Mix macronutrients, micronutrients, vitamins, sucrose, and Agar Agar in distilled water and sterilize it in an autoclave at 121 degrees, 15lbs pressure. Medium Solidification: Pour the nutrient medium into test tubes and allow it to solidify. Seed Sample Preparation: Gather seeds from plants like chili, cotton, corn, rice, etc. Inoculation: Using forceps, transfer each seed sample into the test tubes, ensuring they are placed carefully in the center and partially immersed in the medium. Plug the test tubes with non-absorbent cotton. Incubation: Place the test tubes with the seed samples in a culture growth room and leave them for 7 days to allow germination to occur. Tissue Culture