Team:LACAS BioBots/Procedure
Lab Work
Week 1
In our introduction week, we were given a tour of the laboratory centre where we were introduced to all the equipment and parts of the lab that we’d need for specific purposes. We were shown around the biopharmaceutical facility and learned about various equipment, processes like gel electrophoresis, UV lamps, and warnings related to different substances There we met Sir Moon Sajid who taught us about ethics in the lab, tissue culture, MS(Murashige and Skoog) media, and also introduced us to micro pipetting. He also demonstrated how to weigh the mass of ingredients and how to make percentage solutions. We researched on MS media and its ingredients and their effects on cells as well. After familiarizing ourselves with every nook and cranny of the lab, we then conducted research on SOB(Super Optimal Broth), a nutrient broth which is used for bacterial cultures as well as SOC solution which is SOB but we add 15% glycerol solution to the SOB mix to make it. Agar (7.5g/500ml or 15g/1l of it) is added into the SOC solution to make agar plates for the culture to grow. That week we also created another nutrient broth called LB(Luria Bertani) which is a lysogeny broth and a previous version of SOB.
| Tryptone (formed by milk protein casein | 10 grams |
| Yeast Extract (Insdies of a yeast cell) | 5 grams |
| NaCl (to maintain bacteria's osmotic pressure) | 10 grams |
| Water (to produce Broth) | 1 liter |
| Agar(to turn the media solid so it can be spread into Petri dishes) | 15 grams |
| Tryptone | 20 grams | Yeast Extract | 5 grams | MgCl_2 (1M) | 10 milliliters | NaCl | 2.5 milliliters | MgSO_4 | 10 millilters | Distilled Water | 1 liter |
Week 2
After familiarizing ourselves with the lab, we gathered around ingredients from various parts of lab for the preparation of MS media and autoclaving of water. And so we began the preparation of MS media. Our attempt at making MS media failed because we didn’t take in account that the salts and their anions have to be consistent otherwise they’ll end up reacting with each other therefore rendering the solution useless as it becomes contaminated. However, our second attempt was successful as we learned from our mistake and upon further searching in the inventory, we found pre-prepared Murashige and Skoog salt which upon the addition of a few ingredients, formed the proper MS Media.
| Ammonium Nitrate | 1650 mg/L |
| Calcium Chloride | 44 mg/L |
| Magnesium Sulphate | 370 mg/L |
| Monopotassium Phosphate | 170 mg/L |
| Potassium Nitrate | 1900 mg/L |
| Boric Acid | 6.54 ml/L |
| Cobalt chloride (hydrated) | 0.25 mg/L |
| Ferrous sulphate (hydrated) | 27.8 mg/L |
| Manganese sulfate | 22.3 mg/L |
| Potassium iodide | 0.83 mg/L |
| Sodium molydate | 0.25 mg/L |
| Zinc sulfate | 8.6 mg/L |
| Ethylenediamine tetraacetic acid ferris sodium | 5.57 g – 7.45g |
| Copper sulfate | 0.035 mg/L |
| Myo-inositol | 100mg/L |
| Thiamine.HCl | 1.0 mg/L |
| Glycine | 2.0 mg/L |
| Indole acetic acid | 1 – 30mg/L |
Week 3
We then ordered safflower seeds online and they soon arrived within a week. We tried multiple techniques for growing the seeds, ranging from traditional planting to controlled incubation in the laboratory. Before they were grown the seeds were sterilized using Triton X detergent.
| Triton X-100 (used to remove surface stains present) | 1ml in a 200ml volumetric flask |
| Distilled water | 150-200ml |
The seeds were then prepared for germination. We used sterilized seeds that were surface cleaned with a SDS solution to grow them in controlled environment within the laboratory.To prepare for this, every piece of equipment was autoclaved to minimize contamination, this included filter papers, flasks, petri dishes and distilled water.
Procedure for which is; seeds are washed and placed in water in the small autoclaved flask(40-50ml) and 500microlitres of HgCl solution and 1ml of SDS solution previously prepared are added and it is decanted. Add autoclaved distilled water and decant again until no more bubbles are formed due to the reaction.
| 200ml autoclaved and sterilized conical flasks | 2-3 flasks (one for waste, one for the solutions and one for the sterilization with seeds) |
| Autoclaved filter paper | - |
| Autoclaved distilled water | 200ml |
| HgCl2(Mercurian Chloride) | 5g/100ml |
| SDS (Sodium Dodecyl Sulphate) | 10g/100ml |
The Seeds are now sterilized. The Sterilized seeds(see below) were then sandwiched between wet(autoclaved dist. Water) filter papers and placed in the autoclaved Petri dishes. Multiple Petri dishes with various number of seeds per unit area were set up in the incubator and were monitored and maintained. Most of the seeds had germinated but unfortunately they got contaminated and our samples were infected with fungus.
Week 4
We added agar into SOC so that it could solidify within the petri dishes and carried out inoculation. We conducted a search for Top 10 Cells a group of cells that are resilient, while at the same time are accepting of direct gene editing and DNA manipulation. We went through a series of guides for handling with them. We worked under laminar air hood and use of spirit flame to create an atmosphere with no contamination. We set the plates and carried out streaking and spreading of the top 10 cells.
Week 5
After the streaking process, they’re incubated at 37 Celsius for 16 hours until bacterial colonies are visible. Then Inoculate single colonies and add it to 2ml SOB media and incubate/shake in the incubator shaker for 14 hours. Now we make the 15% glycerol solution (which means 85ml of distilled water and 15ml of glycerol). After this, Flash Freezing is carried out, which is also known as Blast Freezing refers to freezing objects at extremely low temperatures (-80C in this case). This method keeps ice crystals small, which minimizes/prevents moisture loss in the flash frozen object when it thaws. We put cryotubes in ice bucket ( to maintain a low temperature environment for the culture) and add 0.7ml of culture and 0.3ml of glycerol solution (Glycerol is very important to add because it acts as a Cryoprotectant which means that it protects the cells in the culture from the low temperatures and prevents the formed ice crystals from “puncturing” the cell membrane). Freeze to -80C is immediately carried out to preserve the culture.
Week 6
This week we looked into the different uses of SOC and SOB for Transformation.SOC is used for the transformation of competent cells while SOB is used in cultivation of recombinant strains of E. Coli. To make the competent cells, we add 250ml liquid SOB medium + 1ml frozen vial (Let it thaw it a little). After that we Put it in cold sterile environment inside the laminar flow for almost 16 hours. We then take 50ml falcon tubes and pre-chill them and after that we fill them up to a 25ml mark with the incubated 250 ml vial. Next we centrifuge at 3000g/5000RPM at 4C for 10 minute, pellet will form on side and then carefully drain out the supernatant. 20ml of the ice cold CCMB80 buffer was added and swirled carefully to resuspend the pellet and centrifuged again in the same conditions as before. Decant again and added 10 ml CCMB80 again to re suspend, put in vials and falcon tubes and labeled them and kept again in -80C to flash freeze.
We had also finally received our bio brick assembly kit from IGEM administration. We researched our bio brick kit (Structure and positions of our chemicals “map” of the kit and DNA microarrays for characterization etc
Week 7
Week 7 was spent in inoculating our SOC media petri plates with Chloramphenicol antibiotic for Characterization of the blue fluorescence protein DNA. The antibiotic was added for growing the transformed bacteria. A 15 microliter solution of the antibiotic is made and added to 200ml SOC media with agar and poured into plates. Transformation is done by resuspension of the DNA in the selected well in one of the kits plates with 10microlitre distilled H20 in the epindorf. Competent cells are thawed and 50microlitre was pipetted into a 1.5ml cryovial along with the resuspended DNA. They were incubated on ice for 30 minutes and given a heatshock afterwards 950 microlitre SOC was also added to the same 1.5 cryovial. 100microliter of this is inoculated on the set petri dishes with the antibiotic and it is then incubated. Single colonies are then picked and a colony pcr is done however even though we performed the procedure, there were no colonies present.
Safflower Logs
- Thursday, 18th April 2019,
IGEM Lahore team planted safflower seeds:
- 8 cups were filled 1 ½ inches with soil and seeds were placed inside them.
- Water sprayed and plastic wrap tied around with rubber bands.
- Placed near sunlight in a cool area.
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Sunday, 21st April 2019
- 4 of the 8 cups had seeds which began germination namely: Deku, Robspierre the Dumb, Mugetsu and Kakarot.
- All pots sprayed evenly with water.
- Those that germinated kept without plastic wrap, others were kept with it.
- Tuesday, 23rd April 2019
- One new seed has germinated and grown roots: Qliphoth
- Cups were all watered and germinating cups were kept under direct sunlight.
- Seeds that sprouted recently were kept in cool, dry room near sunlight.
- 3 cups remain without any sign of growth and are kept under plastic wraps in a cool room and watered regularly.
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Saturday, 27th April 2019
- 2 plants: Deku and Robspierre have died.
- 3 cups remain without any signs of germination.
- Qliphoth has grown into a shoot and has developed roots.
- Mugetsu and Kakarot are still in shoot form.
- All plants were watered and kept under sunlight.
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Monday, 29th April 2019
- Cutting cups midway from the surface to make sure no roots or sensitive tissue is damaged while extracting germinated plant.
- Drainage holes were made into new pots to drain excess water out and prevent waterlogging and rot.
- New pots were filled halfway with soil.
- Growing shoots were placed inside soil.
- Pots were watered and kept near sunlight. /li>
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Saturday, 4th May 2019
- One of the shoots transferred into a larger pot died.
- Last plant remaining was watered and kept near sunlight.
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Monday, 7th May 2019
- Mugetsu, the last remaining plant has begun drying out.
- Watering is consistent and drainage holes are unclogged.
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Thursday, 9th May 2019
- Mugetsu has died due to dehydration and rot.