Team:NU Kazakhstan/Timeline
May
This month was used for preparation of all equipment, media and reagents that can be necessary for realisation of the project. Also, in May newly recruited and future members of iGEM team acquired skills in basic techniques of biotechnology. The plan of the activities and protocols was worked out in order to keep the big picture of the process and organise the work of all part of the project.
Week 1
24.05.17
- LB broth media and LB agar plates were prepared
- Ampicillin stock was prepared using 1g of antibiotic in 10 ml of dH2O. Stock was stored in the fridge
- Inoculation was performed in 15 ml Falcon tubes, using 5ml LB broth. Single colony was selected from LB agar, the tip was dropped and swirled. Bacterial culture was incubated at 37C in incubator
25.05.17
- Miniprep was performed to extract pJD67 plasmid
- Digestion of pJD67 plasmid was done
- Gel electrophoresis was done using 1.5 % agarose gel, 120V
26.05.17
- TAP media was prepared
27.05.17
- Subculture was done by transferring 100 ul of algae to fresh TAP medium
28.05.17
- Agar washing was done
29.05.17
- OD of growing algae was measured
- Agar washing was done
30.05.17
- OD of growing algae was measured
- Maxiprep was done to extract pJD67 plasmid
- Agar washing was done
31.05.17
- Ethanol precipitation (pJD67) was performed
- TAE buffer, LB broth was prepared
- Agar washing was done using ethanol
- Inoculation of E.coli was done (5 ml of LB broth)
June
In June we focused on collection of the stock DNA which could be used further in electroporations. Miniprep extraction kit seemed to be unpersuasive in its concentrations and we tried to work with Maxiprep with a purpose of obtaining at least 2 ug of plasmid DNA for C.reinhardtii electroporation. At the same time, we used June to develop a biosafety system SuperNova for our project.
1.06.17
- OD of growing algae was measured
- Ampicillin stock was prepared
2.06.17
- Transformation of E.coli was performed using pJD67
- OD was measured
3.06.17
- Inoculation of transformed cells was done
- OD of growing algae was measured
- Miniprep was done, concentration was measured
- Gel electrophoresis was done using 1.5 % gel.
OD
Nanodrop results
5.06.17
- Transformation of E.coli with pHyg3 and pJD67
- Chromium media (0.2mM, 100mM) was prepared
- Agar plates (arg+/hyg+, arg+/ hyg-, arg-/hyg+, arg-/hyg-) were prepared
6.06.17
- Agar plates with ampicillin and amphotericin B were prepared
- Competent cells were prepared
8.06.17
- Transformation of E.coli using pJD67
10.06.17
- Inoculation was done, from liquid media of algae to the plates with ampicillin+amphotericin B
15.06.17
- Starch washing was done, starch was stored in ethanol (+4C)
16.06.17
- Digestion of pHyg3 with HindIII was done
17.06.17
- Inoculation was done for miniprep, to extract pJD67 and pHyg3 plasmids
- Agar washing was done
18.06.17
- Miniprep was done (both pJD67 and pHyg3)
- Concentrations were measure, gel electrophoresis was done
19.06.17
- Digestion of pJD67 was done using HindIII
- Concentration was measured using Nanodrop
- Digestion of pHyg3 with KpnI was done
- Concentration was measured using Nanodrop
20.06.17
- Transfection of algae with pHyg3 and pJD67 was done
21.06.17
- Plating of transfected was done
- Media was prepared
July
Plasmid DNA collection stage proceeded to transformation of C. reinhardtii stage. The Miniprep plasmid extraction procedure was optimized to the Miraprep protocol which appeared to be very efficient in its plasmid DNA concentrations. We tested pHYG3 and pJD67 plasmids in electroporations of algae which gave green colonies and resistance on the selective media. But the main part was still awaiting as the ChrR+Chromodulin genes were still to come.
4.07.17
- Miraprep was done
- pHyg3 plasmid was digested with KpnI
5.07.17
- Concentration of digested sample was measured
6.07.17
- Transformation of algae with pHyg3 was done
8.07.17
- Conducted electroporation of C.reinhardtii with pJD67 plasmid which gives algae an ability to live in non-arginine media
12.07.17
- The plates were overdried and therefore pHYG3 electroporated cells did not grow as it as expected. The colonies were rather formless than round
15.07.17
- Electroporation with pJD67 gave small colonies in no arginine added media
August
In August, we strongly pressed on our Human Practices section. We recruited a school student which was highly interested in biological research and taught how her theoretical knowledge of biology is applicable in the laboratories. Our team also organized a meeting with KazChrome company which is widely involved in the work with hexavalent Chromium and could give a more definitive direction to our project for it to be practically effective in industrial sphere.
18.08.17
- TAP media was prepared
20.08.17
- OD700 and OD750 of algae (for chromium reduction assay) was measured
21.08.17
- Competent cells were prepared
- Inoculation of E.coli (pJD67 plamid) was done in 3 tubes with total volume of 50 ml
- Tubes were incubated at 37 C overnight
21.08.17
- MiraPrep was performed to extract pJD67
- Gel Elctrophoresis after MiraPrep was done using 1% agarose gel, 120 V. Concentration of obtained DNA samples was measured using NanoDrop
- Gel Elctrophoresis
23.08.17
- pJD67 plasmid was digested using HindIII
- Concentration was measured
24.08.17
- CW- algae were electroporated with pJD67 plasmid
- TAP, TAP+arg, TAP+arg+Chr (0.4mM) media were prepared for non-electroporated cells
- TAP, TAP+ Chr (0.2mM), TAP+ Chr (0.4mM), TAP+ Chr (0.6mM), TAP+ Chr (0.8mM) media was prepared for electroporated cells
25.08.17
- 1 ml of electroporated and 1 ml of non-electroporated cells were transferred to assigned media
26.08.17
- Inoculation of E.coli was done
27.08.17
- Competent cells were prepared
- LB broth was prepared
28.08.17
- Transformation of E.coli (dh5α) with pHyg3 (Chr+ChrR)
29.08.17
- Inoculation of E.coli in 50 ml of LB broth with overnight incubation at 37C
30.08.17
- MiraPrep (pHyg3 (Chr+ChrR) was done
- Gel electrophoresis was performed using 1% agarose gel, 120 V
- Concentration of DNA was measured by NanoDrop
31.08.17
- Digestion of pHyg3 (Chr+ChrR) with KpnI
September
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October
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Lorem ipsum dolor sit amet, consectetur adipisicing elit. Aspernatur optio iusto officiis culpa expedita rem nisi repudiandae quas, eveniet, neque nihil pariatur! Doloribus, sunt? Maiores ipsum temporibus consectetur voluptas, placeat perspiciatis officia, distinctio repellat earum.
Lorem ipsum dolor sit amet, consectetur adipisicing elit. Aperiam dolor quas inventore hic delectus, temporibus vel voluptate nemo, repellat eaque nostrum ducimus numquam repudiandae nam. Quibusdam quaerat aspernatur commodi accusantium obcaecati pariatur vel eos quas vero quae rerum nemo nihil non laborum labore magni numquam adipisci voluptatum, voluptates soluta. Vel!
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