Team:ACIBADEM ISTANBUL/Lab
WET LAB
DRY LAB
Table of Contents
SAFETY
Before each lab session, we would have a meeting in a recreation room, dubbed by us as the, “iGEM room” where we would:
2) Conduct a dry run of the days protocol
3) listing and reserving the equipment that we shall be using for that day,
4) listing and locating the supplies needed
5) The estimated completion time
6) The most critical steps where we should be extra cautious.
7) Highlight the risk factors at play as well as the respective safety precautions needed.
Risk Factors and Precautions
To summarize however, there is only one critical risk factor to consider, that being the Crotalus atrox snakes venom.
The Crotalus atrox snake venom is a toxic substance and may cause acute toxicity if inhaled (Category 1), swallowed (Category 2) or touched (Category 2) without appropriate protection.
The precautions that we took were:
- Wearing gloves before handling.
- Thoroughly washing hands after handling.
- Notified the Schools doctor about our use of a hemotoxic venom, in case an accident were to take place.
Animal Use
An ex vivo experiment was attempted utilizing the descending aorta from a bovine with……………….. results.
Lab Rules and Training
Lab coats should be worn at all times in the laboratory and should be taken off before leaving.
Close toed shoes should be worn in the lab at all times.
Students must come to the lab with long pants.
Gloves should be worn at all times, with the only exception being when handling high temperature apparatus, in which oven mitts should be worn.
General lab culture
Schedule the use of a common equipment at least a day before hand.
Prepare your rack and other equipment before hand.
Always ask before using something.
If you take something, put it back in its original place (and note the amount taken if required).
Treat everyone and everything with respect.
No eating/drinking in the lab under any cricumstances.
If you don’t know the answer to something, don’t be afraid to ask.
If something breaks/malfunctions, inform the lab supervisor immediately.
Labelling
Always use permanent marker to label.
Labelling: Label your equipment, reagents and disposables in this format:
Name of Specimen / Lab Name / Your Name / Date
Autoclaving
Before autoclaving, ask the other laboratory staff if they have anything that needs to be autoclaved.
Glassware: Remove previous autoclave tapes; remove any labelling with 70% Alcohol.
Labels should be made on top of the autoclave tape itself.
If it has a cap: loosely close it so that the pressure can pass into it.
Cover beakers and Erlenmeyer flasks with two layers of aluminum foil.
Pipette tips and microfuge tubes: If sterile, cover the mouth of the container with autoclave tape.
If not sterile, place autoclave tape on top of the container.
Bacterial culture
Bacteria stock should always be stored in +4c.
When labelling your petri plates, do not forget to write the Species and Strain name.
Inspect colony shape, size and color to ensure no contamination.
Always pick up single colonies to ensure purity.
The top on the petri plate should always be placed upside down.
Wrap the preti plates in paraffin to avoid contamination and make it safer to handle.
Interlab
To InterLabHemolytic Activity Assay
| What is haemolysis Hemolysis or haemolysis also known by several other names, is the rupturing (lysis) of red blood cells (erythrocytes) and the release of their contents (cytoplasm) into surrounding fluid (e.g. blood plasma). Why haemolysis test Haemolysis test is a must, when a new molecule is intended for human use as it gives us invaluable data about it’s toxicity. How does it work A red blood cell solution is prepared and incubated with different concentrations of the peptide in question. Then the haemolytic rate is calculated according to results of the spectroscopic measurements which are taken at 415 nm, the wavelength haemoglobin gives it’s peak. Different concentrations used in protocol gives us valuable data about it’s dose related toxicity. |
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| PBS (Phosphate Buffered Saline) Triton (1x will be used but having a 10x stock is beneficial) MilliQ Water Red Blood Cell Solution 0.25% (RBC solution) Samples [For ours they are 10mg/mL (~9.26 mM), 1 mg/mL (926 uM), 0.1 mg/mL (92.6 uM), 0.01 mg/mL (9.26 uM), 0.001 mg/mL (926 nM) However 300 uM is the advised max concentration] |
| Samples : 20 uL test sample + 100 uL RBC solution Controls: 20 uL triton + 100 uL RBC solution / 20 uL PBS + 100 uL RBC solution Samples and controls were incubated at 37C for 1 hour. After incubation, 100 uL of supernatant per sample (without disturbing the pellet) were taken to flat bottom plate for measurement. Spectrophotometer measurements were taken at 415 and 600 nm. |
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| According to our results, LTNF 2.0 does not have significant haemorrhaegic effect, even at higher doses. |
| Derivative A ---- LTNF 10 (10 aminoacid sequence of original LTNF which shows activity) Derivative B ----- LTNF 10 + 10 (10 additional aminoacids were added and cysteine was added to the beginning and the end of the sequence) Derivative C-------- LTNF 10 + 10 (10 additional aminoacids were added and cysteine was added to the beginning and the end of the sequence. The peptide was circularized.) |
HPLC Peptide Stability
| What is HPLC High Performance Liquid Chromatography (HPLC) is a type of column chromatography that pumps a sample mixture or analyte in a solvent (known as the mobile phase) at high pressure through a column with chromatographic packing material (known as the stationary phase). HPLC has the ability to separate, and identify compounds that are present in any sample that can be dissolved in a liquid in trace concentrations. Because of this versatility, HPLC is used in a variety of applications, such as pharmaceutical, environmental, forensics, and chemicals. Sample retention time will vary depending on the interaction between the stationary phase, the molecules being analyzed, and the solvent, or solvents used. As the sample passes through the column it interacts between the two phases at different rate, primarily due to different polarities in the analytes. Why HPLC We are using HPLC because of it’s sensitivity and accuracy.We are capable of measuring the metabolites at very little concentrations. (Almost as soon as they appear) How does this work We are incubating our peptide in different intervals in human plasma which is seperated from it’s fatty content and at 37 C creating the optimum environment for mimicing human body. The peptide acts as if it is in the body. After appropriate amount of time has passed, the sample is taken in to HPLC for measurement. As soon as any metabolite appear, the sensitivity of HPLC allows us to observe it as a peak and gives us data about it’s amount and polarity. |
| HPLC Eppendorf Tubes Floating foam rack Water Bath Vortex Fridge |
| Sample Pure water Human serum 6M Urea solution TCA: 20% (v/v) |
| CALIBRATION STEP PSS B for every peptide is loaded into the HPLC (100 uL is taken in vial but 90 uL is used). (40 ul PSS B + 80 ul pure water) ANALYSIS STEP Peptide/serum solution is seperated into 3 equal parts (813/3 = 271 uL) The solutions are vortexed and 40 uL is taken to an eppendorf tube Rest of the solution is taken into water bath to incubate at 37 C (Parafin the caps) 40 uL Urea solution is added on the 40 uL stock solution The urea-stock mix is vortexed and taken to fridge Wait 10 minutes Add 40 uL TCA , vortex and take it to fridge Wait 10 min Santifruge (10 min / 15000 rpm) Take 100 uL supernatant (DONT TOUCH THE PELLET) Put it into the insert HPLC measurement. (90 uL will be used for measurement) (Blue writings are done for all measurements. As Rest of the stock is incubated according to protocol (for us 0,1,2,4,8,24 hr measurements) After taken from incubation, santifruge the stock peptide/serum stock at 15000 rpm for 2 minutes |
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| According to our results, LTNF 2.0 is more stable in blood stream compared to the native LTNF 10. |
| Derivative A ----- LTNF 10 (10 aminoacid sequence of original LTNF which shows activity) Derivative B ----- LTNF 10 + 10 (10 additional aminoacids were added and cysteine was added to the beginning and the end of the sequence) Derivative C----- LTNF 10 + 10 (10 additional aminoacids were added and cysteine was added to the beginning and the end of the sequence. The peptide was circularized.) |
Vascular Ex-vivo Assay
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deserunt ipsum! Lorem ipsum dolor sit amet, consectetur adipisicing elit. Mollitia autem quasi inventore unde nobis voluptatibus illum quae rerum laudantium
minima, excepturi quis maiores. Eaque quae, nam delectus explicabo, deserunt ipsum!
Lorem ipsum dolor sit amet, consectetur adipisicing elit. Mollitia autem quasi inventore unde nobis voluptatibus illum quae rerum laudantium minima, excepturi quis maiores. Eaque quae, nam delectus explicabo,
deserunt ipsum! Lorem ipsum dolor sit amet, consectetur adipisicing elit. Mollitia autem quasi inventore unde nobis voluptatibus illum quae rerum laudantium
minima, excepturi quis maiores. Eaque quae, nam delectus explicabo, deserunt ipsum!
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deserunt ipsum! Lorem ipsum dolor sit amet, consectetur adipisicing elit. Mollitia autem quasi inventore unde nobis voluptatibus illum quae rerum laudantium
minima, excepturi quis maiores. Eaque quae, nam delectus explicabo, deserunt ipsum!
Lorem ipsum dolor sit amet, consectetur adipisicing elit. Mollitia autem quasi inventore unde nobis voluptatibus illum quae rerum laudantium minima, excepturi quis maiores. Eaque quae, nam delectus explicabo,
deserunt ipsum! Lorem ipsum dolor sit amet, consectetur adipisicing elit. Mollitia autem quasi inventore unde nobis voluptatibus illum quae rerum laudantium
minima, excepturi quis maiores. Eaque quae, nam delectus explicabo, deserunt ipsum!
Lorem ipsum dolor sit amet, consectetur adipisicing elit. Mollitia autem quasi inventore unde nobis voluptatibus illum quae rerum laudantium minima, excepturi quis maiores. Eaque quae, nam delectus explicabo,
deserunt ipsum! Lorem ipsum dolor sit amet, consectetur adipisicing elit. Mollitia autem quasi inventore unde nobis voluptatibus illum quae rerum laudantium
minima, excepturi quis maiores. Eaque quae, nam delectus explicabo, deserunt ipsum!
Gel Degredation ELISA
| What is ELISA The enzyme-linked immunosorbent assay (ELISA) is a frequently used analytical biochemistry assay. The assay uses a solid-phase enzyme immunoassay (EIA) to detect the presence of a ligand (commonly a protein) in a liquid sample using antibodies. The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality-control check in various industries. Antigens from the sample are attached to a surface. Then, a further specific antibody is applied over the surface so it can bind to the antigen. This antibody is linked to an enzyme, and, in the final step, a substance containing the enzyme's substrate is added. The subsequent reaction produces a detectable signal, most commonly a color change in the substrate. Why ELISA ELISA is picked because of it’s specificity. Type IV collagen is used as antigen. One of the key event (maybe the key event) of the onset of haemorrhagic activity is type IV collagen degredation. How does it work Gelatine, hydrolized Type IV collagen, are attached to the bottom of the well. Then, they are incubated with samples (which are venom, venom+protein,blank etc.) Main mechanism of the toxicity of the venom is it’s haemorrhagic activity, which is mainly caused by type IV collagen degredation in basement membrane of the vessels (particularly capillaries) Thus, we expect venom to degredate the gelatine when given on its own and to see no gelatinolytic activity when given with our peptide. Then we add anti-gelatin serum ,capable of binding to gelatin. When we add the substrate which is capable of binding the enzymes in the anti-gelatin serum, it ‘s colour intensity can be measured at 405 nm. |
| ELISA Plate Centifruge Spectrophotometer Eppendorf Tubes Micropipettes Falcon Tubes |
| Venom Bovine Gelatine PBS Rabbit antigelatin serum Anti-rabbit Ig G alkaline phosphatase Sigma 104 phosphatase substrate Glycine Buffer Tris CaCl2 Brij Samples (Peptide) |
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Modelling
To ModelPlasmid Design
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deserunt ipsum! Lorem ipsum dolor sit amet, consectetur adipisicing elit. Mollitia autem quasi inventore unde nobis voluptatibus illum quae rerum laudantium
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