1. With a pipette tip, punch a hole through the foil cover into the corresponding well of the part.
2. Pipette 10µL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended.
3. The resuspension will be red, as the dried DNA has cresol red dye.
2. Weigh 0.25-0.5 g of Chloramphenicol;
3. Add 10 ml of 70% EtOH;
4. Dissolve completely;
5. Stock may be kept at -20°C for 1 year.
1. Fill the ice bucket with ice, and pre-chill 1.5ml tubes.
2. Thaw competent cells on ice: This may take 10-15min for a 260µl stock. Dispose of unused competent cells. Do not refreeze unused thawed cells, as it will drastically reduce transformation efficiency.
3. Pipette 50µl of competent cells into 1.5ml tube: 50µl in a 1.5ml tube per transformation.
4. Pipette 1µl of resuspended DNA into 1.5ml tube. Gently pipette up and down a few times. Keep all tubes on ice.
5. Pipette 1µl of control DNA into 2ml tube: Pipette 1µl of 10pg/µl control into your control transformation. Gently pipette up and down a few times. Keep all tubes on ice.
6. Close 1.5ml tubes, incubate on ice for 30min.
7. Heat shock tubes at 42°C for 45 sec: 1.5ml tubes should be in a floating foam tube rack. Timing is critical.
8. Incubate on ice for 5min.
9. Pipette 950µl SOC media to each transformation: SOC should be stored at 4°C, but can be warmed to room temperature before use.
10. Incubate at 37°C for 1 hour, shaking at 200-300rpm.
11. Pipette 100µL of each transformation onto petri plates Spread with sterilized spreader or glass beads immediately. This helps ensure that you will be able to pick out a single colony.
12. Weigh 0.25g of Chloramphenicol and add 10 ml of 70% EtOH. Dissolve completely.
13. Pipette 25 mL of LB broth onto petri plates Spread with sterilized spreader or glass beads immediately. This helps ensure that you will be able to pick out a single colony.
14. Incubate transformations overnight (14-18hr) at 37°C: Incubate the plates upside down (agar side up).
1. Spin the cell culture in a centrifuge to pellet the cells, empty the supernatant (media) into a waste collection container.
2. Resuspend pelleted bacterial cells in 250 µl Buffer P1 (kept at 4 °C) and transfer to a microcentrifuge tube. No cell clumps should be visible after resuspension of the pellet. Important: Ensure that RNase A has been added to Buffer P1.
3. Add 250 μl Buffer P2 and gently invert the tube 4–6 times to mix. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min.
4. Add 350 μl Buffer N3 and invert the tube immediately and gently 4–6 times. To avoid localized precipitation, mix the solution gently but thoroughly, immediately after addition of Buffer N3. The solution should become cloudy.
5. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge. A white pellet will form.
6. Apply the supernatants from step 4 to the QIAprep spin column by decanting or pipetting.
7. Centrifuge for 30–60 s. Discard the flow-through.
8. Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60s. Spinning for 60 seconds produces good results.
10. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.
1. If the protein is expressed
a. Induce the cells containing IsPETase gene controlled by T7 promoter with 0.4M, 0.1M, or 0M IPTG for either 6 hours or overnight, at either 15- or 30-degrees C.
b. Expression of T7 Cells
c. Lysis of cells according to our ultrasonication protocol.
d. Validate protein expression further with SDS Page (An SDS Page will confirm by showing that a protein of the correct weight is being expressed)
e. Agarose gel electrophoresis.
f. Histag purification.
g. Western blotting (A Western blot will show that our histag is functioning);
h. P-nitrophenyl butyrate (pNPB) Assay
2. NO – reperform the transformation.
3. Transformation of MHETase to Pseudomonas putida KT2440 with terminator T7 and promoter J23106.
4. Validate protein expression further with SDS Page.
5. PCR (to show contained in the cells by band size (1813 bp) in electrophoresis gel);
Having had restricted access to the lab space and materials, we could not accomplish everything we planned. Despite our setbacks, we made a huge effort to carry out as many experiments as possible as mentioned in our lab notebook.
Our primary goal was to show that P. putida KT2440 could gain a new function by incorporating a foreign gene coding for PETase enzyme. We performed experiments according to the plan, and we were able to show that P. putida KT2440 strain is able to get transformed (Picture 1). The next experimental steps were halted due to timing and lab space constraints, but we developed a plan of future experiments to prove our theory. According to our plan we will continue our work with SDS Page and Western blotting, showing that a protein of the correct weight will be expressed. After we planned to transform MHETase enzyme to Pseudomonas putida KT2440 with terminator T7 and promoter J23106. So, in the end these enzymes, by working in pairs form SUPER ENZYME, which is able to accelerate the decomposition of the plastic process several times and convert it efficiently into its two environmentally benign monomers.
References:
Biosafety Guidelines for Handling Microorganisms in the Teaching Laboratory: Development and Rationale by: Elizabeth A. B. Emmert and the ASM Task Committee on Laboratory Biosafety Department of Biological Sciences, Salisbury University, Salisbury. JOURNAL OF MICROBIOLOGY & BIOLOGY EDUCATION, May 2013, p. 78-83 Safety
Pseudomonas putida KT2440 (ID 139665) - BioProject - NCBI (nih.gov)
Structural studies reveal the molecular mechanism of PETase - PubMed (nih.gov)
Antibiotic Usage | Herman Lab | Nebraska (unl.edu)
https://www.protocols.io/view/How-to-make-a-25-50-mg-ml-Chloramphenicol-Stock-So-k3byl4rjgo5d/v1
Part:BBa K2010000 - parts.igem.org
Team:Harvard BioDesign/Experiments - 2016.igem.org
Конструирование и клонирование разлагающих пластик рекомбинантных ферментов (MHETase) - PubMed (nih.gov)