Team:NU Kazakhstan/Notebook
Notebook
Project Timeline
| Week | Dates | Work completed |
|---|---|---|
| 1 | May 1-5 | - Lab tools and reagents availability check-up -Measurement of Gibco BG-11’s pH |
| 2 | May 6-12 | - Laboratory introduction & safety training for new team members - Agar plates preparation according to LB agar protocol - E.coli (DH5ɑ) streaking on LB agar and incubation - LB broth media preparation according to general E.coli protocol - Grown E.coli transfer to LB broth and incubation - Competent cells preparation according to preparing competent cells protocol - SOC media (pH=7.18) preparation according to the protocol - Chemical transformation of competent DH5ɑ cells and streaking on LB agar media |
| 3 | May 13-19 | - Miraprep of cultures according to miraprep protocol - Cyanobacteria subculturing - Agarose gel preparation with 10l/100 ml Sybr Safe addition - Gel electrophoresis of miraprep products - Measuring the concentration according to Nanodrop protocol - Dilution of primers - PCR of liquid chlamy cultures according to liquid PCR protocol - DNA extraction from SQR- cyanobacteria cultures according to the DNA extraction protocol - Amplification of the extracted DNA (PCR) - HydA amplification from the PCR products - Gel electrophoresis of PCR products - Nanodrop of the products - Hydrogenase construct (joined Hyd genes) creation: PCR amplification of Hyd1, Hyd2 and Hyd3 separately - Check of PCR products via gel electrophoresis |
| 4 | May 20-26 | - Circular polymerase extension cloning (CPEC) (pSyn6+hydrogenase) assembly - PCR and gel electrophoresis - Cyanobacteria subculturing - NS site amplification via PCR - Gel electrophoresis of the PCR product - Nanodrop of the PCR products - Chemical transformation of NS site into E.coli |
| 5 | May 27-June 2 |
- DNA extraction from old cyanobacteria cultures - PCR of hydrogenase construct parts: Hyd1, Hyd2, Hyd3 - Check of SQR gene presence in cyanobacteria via PCR - Gel electrophoresis of the PCR products - 4 LB agar plates: 2 with and 2 without spectinomycin preparation - DH5ɑ streaking on the plates and incubation - LB broth tubes preparation and transfer of E.coli from agar plates and incubation - OD measurement of the DH5ɑ cultures via Spectrophotometry - Competent cells preparation - Transformation of Hydrogenase construct into the E.coli with higher OD readings - Plating transformed cultures on spectinomycin plates - OD measurement of old cyanobacteria cultures - Cyanobacteria subculturing - Transformation of cyanobacteria according to Invitrogen protocol - CPEC (pSyn6+hydrogenase) assembly - Addition of Ascorbic Acid to C, 58B + Cr, WT + Cr Chlamy cultures - Preparation of chromium and Ascorbic Acid solutions in TAP S- - Preparation of the solution with ascorbic acid + chromium in absolute ethanol |
| 6 | June 3-9 |
- NaHCO3stock solution preparation - pH calibration of sodium thiosulfate, NaHCO3and BG-11 via 37% HCl - BG-11 agar plates with NaHCO3and spectinomycin preparation according to Preparation BG-11 plates protocol - Gel extraction according BioRad protocol for obtaining pSyn6 and CPEC pure DNA samples - Transformation of DNA into E.coli - OD measurement of the samples - Miraprep of the samples - Inoculation of cyanobacteria - Transformation of cyanobacteria according to Stony Brook team protocol - Preparation of BG11 agar plates - Liquid PCR - Nanodrop - Gel electrophoresis - DNA purification according to Monarch purification kit protocol - Nanodrop of the purified samples |
| 7 | June 10-16 |
- Re-dilution of stock Hyd samples - Amplification of Hyd1, Hyd2 and Hyd3 via new PCR protocols - Samples purification according to Monarch purification kit protocol - Nanodrop and gel electrophoresis of the samples - CPEC samples amplification - Transformation into DH5ɑ strain - Transfer of cultures from agar plates into LB broth media - Measurement of absorbance and collection of obtained UV graphs of carbon quantum dots - Liquid PCR - Gel electrophoresis |
| 8 | June 17-23 |
- Miraprep of transformed culture - Purification of miraprep samples - Miraprep samples DNA linearization according to Restriction digestion protocol - Plasmid to insert ratio recalculation for CPEC - CPEC (pSyn6+hydrogenase) assembly - Liquid PCR - Gel electrophoresis - Dilution of primers - HydA amplification via PCR - Check via nanodrop and gel electrophoresis |
| 9 | June 24-30 |
- Mixing with ZnCl2 and burning in furnaces of two cyanobacteria samples (sulfide and control) - Washing with HNO3 and HCl, filtering and drying two cyanobacteria samples (sulfide and control one) - PAM stock dilution - HydA amplification from PAM - Liquid PCR - Gel electrophoresis - Preparation of TAP media - Analysis of the prepared carbon dots |
| 10 | July 1-7 |
- Check of reagents presence for BG-11 media preparation - Linearization of PAM HydA and p233 Maturation factor (MF) DNA by BAM-HI - Gel electrophoresis check - Psba1 amplification - CPEC (pSyn6+hydrogenase) assembly - Verification of insert integration into the vector through amplification of region - Linearization of CPEC DNA - Nanodrop and gel electrophoresis - Liquid PCR - Gel electrophoresis |
| 11 | July 8-14 |
- Polymerase cycling assembly (PCA) creation - Miraprep - Nanodrop and gel electrophoresis - Preparation of the TAP media - Liquid PCR - Gel electrophoresis |
| 12 | July 15-21 |
- Gel electrophoresis - Hydrogenase construct assembly - CPEC (pSyn6+hydrogenase) assembly - Linearization of CPEC sample - Nanodrop and gel electrophoresis of CPEC samples - Purification of psba1 gene - HydA amplification from PAM - Transformation of CPEC - Liquid PCR - Subculturing |
| 13 | July 22-28 |
- Subculturing of cultures from agar plates into LB broth - Check for the contamination in the reactants - Nanodrop - Gel electrophoresis - PCR - Enzyme digestion of CPEC miraprep samples - HydA and PsbA1 assembly via Polymerase Cycling Assembly (PCA) |
| 14 | July 29- August 4 |
- MF plasmid check via nanodrop - Linearization of MF plasmid - Gel electrophoresis - HydA and PsbA1 assembly via Polymerase Cycling Assembly (PCA) |
| 15 | August 5-11 |
- Primers redesigning - Cleaning lab - Subculturing of cultures |
| 16 | August 12-18 | - Preparation of ingredients for the reduction assay |
| 17 | August 19-25 |
- Preparation of chromium reduction assay - Liquid PCR for the presence of SQR - Filter paper preparation - Latex’s pH measurement |
| 18 | August 26 - September 1 |
- Dilution of primers - Amplification of Hyd 1, Hyd2 and Hyd3 - Purification of Hyd DNAs according to Monarch protocol - Nanodrop - Hydrogenase assembly via Polymerase Cycling Assembly (PCA) from Hyd1, Hyd2 and Hyd3 - Amplification of obtained PCA samples - Subculturing from liquid culture of cyanobacteria to latex on filter paper - Rhodopsin amplification - Gel electrophoresis - Overlaps (to Hyd1and pSyn6) addition to SQR gene -pSyn6 digestion with Kpn and BgL-II restriction enzymes |
| 19 | September 2-8 |
- Hyd1 and Hyd3 amplification - Gel electrophoresis - Hydrogenase assembly via Polymerase Cycling Assembly (PCA) from Hyd1, Hyd2 and Hyd3 - Amplification of obtained PCA samples - Insert (Hydrogenase construct) integration into pSyn6 vector - Survivability of cyanobacteria assay on latex covered filter papers - Purification of Hyd1, Hyd2, Hyd3 according to Monarch purification kit protocol - Assembly (PCA) from Hyd1, Hyd2 and Hyd3 with a new approach - Amplification of obtained PCA samples - Gel electrophoresis check of PCA samples - Hyd2 amplification with extended cycles of PCR |
| 20 | September 9-15 |
- Purification of Hyd1, Hyd2, Hyd3 with nuclease free water - Amplification of Hyd1, Hyd2 and Hyd3 with a new Master Mix Phusion for the high GC content - Gel extraction according to Invitrogen Gel Extraction kit protocol - Nanodrop |
| 21 | September 16-22 |
- Insertion of psba1+HydA into MF vector - HydA and psba1 amplification - Purification of the amplified samples - Insert and MF digestion with BamHI-HF - Purification of the digested samples - Ligation of insert and MF - Amplification of construct using E.coli - Transformation of construct into cyanobacteria |
| 22 | September 23-29 |
- Ligation of insert and MF - H2S Survivability assay of cyanobacteria - Transformation of construct into cyanobacteria |
| 23 | September 30- October 6 |
- Preparation of liquid cultures with/without chloramphenicol antibiotic - Work on characterization part: mRFP generator (Part:BBa_K861173) - Transformation of mRFP generator into E.coli - Nanodrop - Gel electrophoresis of the samples - Transformation into BL21 (DE3) according to the BioBrick part protocol - H2S Survivability assay of cyanobacteria - Preparation of the TAP media |
| 24 | October 7-13 |
- HydA amplification from the miraprep samples - H2S Survivability assay of cyanobacteria |
| 25 | October 14-20 |
- H2S Survivability assay of cyanobacteria - HydA amplification from the miraprep samples |
| 26 | October 21-22 |
- Transformation of construct into cyanobacteria - Hydrogen production assay |