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.
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
In September, the genes from IDT and pHYG3 ChrR+Chromodulin were on our hands. Transcriptional units from IDT were ligated with pChlamy_4 vector which possesses an endogenous promoter for C. reinhardtii and makes transformation more effective. At the same time pHyg3(Chr+ChrR) was electroporated into algae to test it further for expression of Chromate Reductase/Chromodulin proteins and actual reduction of hexavalent chromium.
1.09.17
- Transformation of E.coli with pHyg3(Chr+ChrR) was performed
2.09.17
- Inoculation of E.coli in 50 ml of LB broth with overnight incubation at 37C was done
3.09.17
- Miraprep was done to extract pHyg3(Chr+ChrR) plasmid
- Concentration was measured using NanoDrop
- Gel electrophoresis was done using 1% gel, 120V
4.09.17
- pHyg3(Chr+ChrR) plasmid was linearized
- Concentration was measured using NanoDrop
- Gel electrophoresis was done using 1% gel, 120V
5.09.17
- Agar washing was done
- TAP agar plates were prepared for electroporated cells: TAP Hyg+ 0mM Cr, TAP Hyg+ 0.05 mM Cr, TAP Hyg+ 0.1 mM Cr, TAP Hyg+ 0.2 mM Cr
- TAP agar plates for non-electroporated controls: TAP Hyg- 0 mM Cr,TAP Hyg- 0.05 mM Cr, TAP Hyg- 0.1 mM Cr, TAP Hyg- 0.2 mM Cr
- Agar plates left aging in the Biosafety Cabinet for several days (12 hours-4 days to ensure sterile conditions of the hood)
6.09.17
- Agar washing was done
- Agar plates left aging in the Biosafety Cabinet for several days (12 hours-4 days to ensure sterile conditions of the hood)
7.09.17
- Transformation of algae with pHyg3(Chr+ChrR) - electroporation
- Agar plates left aging in the Biosafety Cabinet for several days (12 hours-4 days to ensure sterile conditions of the hood)
8.09.17
- Overnight incubation of electroporated cells and plating on Hyg+ TAP plates with different Chromium concentration: 0mM, 0.05 mM, 0.1 mM, 0.2 mM
- Control non-electroporated cells are following the same protocol as well as electroporated cells and plated on non-antibiotic selective media:TAP Hyg- 0 mM Cr,TAP Hyg- 0.05 mM Cr, TAP Hyg- 0.1 mM Cr, TAP Hyg- 0.2 mM Cr
9.09.17 - 14.09.17
- Agar washing
- 4-7 days waiting period for growth of electroporated algae colonies on the Hyg+ plates
15.09.17
- Electroporation of Chlamydomonas reinhardtii with pHYG(Chr+ChrR)
- Overnight incubation
- Chromate reduction assay subculturing for optimal OD 1.2
16.09.17
- Plating of electroporated algae on TAP minimal media (without acetate) which reinforces C.reinhardtii to undergo only photosynthetic nutrition: TAP min Hyg+ 0 mM Cr,TAP min Hyg+ 0.05 mM Cr, TAP min Hyg+ 0.1 mM Cr, TAP min Hyg+ 0.2 mM Cr
- Non-electroporated control plates: TAP min Hyg- 0 mM Cr, TAP min Hyg- 0.05 mM Cr, TAP min Hyg- 0.1 mM Cr, TAP min Hyg- 0.2 mM Cr
17.09.17
- Observation of the growth of algae on TAP min plates for 7-10 days (takes longer time due to lack of acetate
- Opening and wrapping the TAP min plates every 14 hours and 10 hours because growth of algae on TAP min plates requires photoperiodic phases of 14 and 10 hours relatively in the light and darkness for 10 days
19.09.17
- Gene arrival from IDT: SuperNova, ChrR and Chromodulin
- OOpening of electroporated cells at 12 am, closing at 10 pm
20.09.17
- PCR of transcriptional units from IDT didn’t work because of low quality DNA of ChrR and Chromodulin design synthesis. PCR of SuperNova showed a good result at the band of 800-900 bp
21.09.17
- Chromate reduction assay from electropporated colonies: measurements of chromium concentration every 2 hours
- Optimization of PCR. PCR of transcriptional units of ChrR, SuperNova and Chromodulin were amplified from pHYG(ChrR+Chromodulin)
23.09.17
- Ligation of amplified transcriptional units with pChlamy_4 vector
- Chromate reduction assay chromium measurements w=in the electroporated cells do not show the change in the concentration
24.09.17
- Transformation of ligated pChlamy_4 with transcriptional unit of ChrR into the competent E.coli and plating on ampicillin plates.
25.09.17
- Inoculation of single colony to 50 ml culture for Miraprep
- Chromate reduction assay: standard curve is recalculated
26.09.17
- pChlamy_4+ChrR plasmid extraction by Miraprep protocol
- Digestion with ScaI restriction endonuclease
- Chromium concentration measured in the liquid electroporated culture
27.09.17
- Preparation of Zeocin+ TAP agar plates which are left for aging for 4 days
- Preparation of TAP min Hyg+ 0 mM Cr,TAP min Hyg+ 0.05 mM Cr, TAP min Hyg+ 0.1 mM Cr, TAP min Hyg+ 0.2 mM Cr for electroporation of pHYG3 ChrR+Chr
- Non-electroporated control plates: TAP min Hyg- 0 mM Cr, TAP min Hyg- 0.05 mM Cr, TAP min Hyg- 0.1 mM Cr, TAP min Hyg- 0.2 mM Cr (left for aging)
- Sonication of liquid cultures. Chromate reduction assay measures the concentrations of sonicated cell which reveals the amount of inner chromium concentration
October
October was busy with characterization of transcriptional units through the PCR colony procedure to ensure expression of Chromate reductase in transformed algae. Electroporated colonies were transferred to liquid cultures to conduct a series of chromate reduction assays. Meanwhile, new electroporations are conducted to replenish the number of transformed algal colonies. Getting prepared for submission of the results!
2.10.17
- Electroporation of pHYG3 ChrR+Chr into C.reinhardtii with overnight incubation
- Plating of electroporated pChlamy_4 ChrR on Zeo+ plates
- Inoculation of colonies from different plates of electroporated algae to liquid culture for chromium concentrations measurements
3.10.17
- Plating of electroporated pHYG3 ChrR + Chr on Hyg
- Chromate reduction assay measurements
4.10.17
- Ligation of SN transcriptional unit into pChlamy_4 plasmid
- Transformation into competent E.coli and plating on Amp+ plates
5.10.17
- Inoculation of single colony to 50 ml culture for Miraprep
6.10.17
- Miraprep protocol pChlamy_4 + SN extraction
- Restriction digestion with ScaI endonuclease
- PCR Purification of linear plasmid
- Chromate reduction assay measurements
7.10.17
- Electroporation of pChlamy_4 + ChrR and pChlamy_4 + SN into C.reinhardtii with overnight incubation
- pChlamy_4 + SN is constantly wrapped in foil to prevent activation of SuperNova under 500-600 nm wavelenght light
- Greeen colonies appeared on plates TAP agar minimal with electroporated pHYG3 ChrR + Chr
10.10.17
- Green colonies observed for pHYG3 ChrR + Chr electroporated cells after 7 days
- Colony PCR showed red bands under UV light
12.10.17
- Arrival of primers for parts submission so started with PCR of transcriptional units of promoter, terminator and SN from SN transcriptional unit IDT, Chromodulin and ChrR from pHYG3 ChrR+Chr and pSB1C3 plasmid backbone
- PCR Purification clean up of amplified transcription units
13.10.17
- CPEC ligation protocol failed due to fast-ramp mode of Thermocycler
- Colony PCR still shows red bands under UV light
15.10.17
- CPEC is successful for terminator and SuperNova
- PCR purification of ligated parts
- Transformation of CPEC ligated parts into competent E.coli
- Chromate reduction assay does not show any substantial change in the inner and outer concentration of chromium in the electroporated cultures
16.10.17
- Inoculation of single colony into 50 ml cultures
- Colony PCR is repeated to eliminate the source of red bands and ensure the presence of the parts in transformed algae
17.10.17
- pSB1C3 + term and pSB1C3 + SN plasmid extraction using Miraprep protocol
- Colonies observed for electroporated cells pChlamy_4 ChrR
18.10.17
- CPEC successful for promoter, ChrR and Chromodulin
- PCR purification of ligated parts and transformation into competent E.coli
19.10.17
- Inoculation into liquid culture of 50 ml
- Colony PCR and silver staining of DNA PAGE
- Chromate reduction assay has positive results of chromium concentration change
20.10.17
- Miraprep extraction of parts pSB1C3 + prom, pSB1C3 + ChrR and pSB1C3 + Chr
- Drying and preparation for submission of parts
- Colony PCR did not show the bands
- DNA PAGE Silver staining
22.10.17
- Colony PCR according to new protocol did not work out.
23.10.17-25.10.17
- We repeated colony PCR and PCR on liquid cultures. We successfully amplified chromate reductase.
26.10.17-31.10.17
- Repeated chromium reduction assay to verify results.
