Team:Hannover/Notebook/Heavy Metal
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- | <h1>Notebook / Heavy metal</h1> | + | <h1><a href"https://2014.igem.org/Team:Hannover/Notebook">Notebook</a> / Heavy metal</h1> |
- | <p class="text">Here you can find a list of lab work concerning our main project "heavy metals". This includes the stable transformation of <i>Arabidopsis thaliana</i> (<i>A. thaliana</i>), the heterologous expression and the quantitative analysis of our T4MBP.</p> | + | <p class="text">Here you can find a list of lab work concerning our main project "heavy metals". This includes the stable transformation of <i>Arabidopsis thaliana</i> (<i>A. thaliana</i>), the heterologous expression and the quantitative analysis of our T4MBP.<br><br>ONC = overnight culture ; CBD = Cellulose binding domain ; IPG = Institute for plant genetics ; OD = optical density ; PCR = Polymerase chain reaction ; RT = room temperature ; T4MBP = Top 4 metal binding protein ; ICP-OES = inductively coupled plasma optical emission spectrometry ; ICP-MS = inductively coupled plasma mass spectrometry ; MS = mass spectrometry</p> |
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Latest revision as of 13:29, 17 October 2014
Notebook / Heavy metal
Here you can find a list of lab work concerning our main project "heavy metals". This includes the stable transformation of Arabidopsis thaliana (A. thaliana), the heterologous expression and the quantitative analysis of our T4MBP.
ONC = overnight culture ; CBD = Cellulose binding domain ; IPG = Institute for plant genetics ; OD = optical density ; PCR = Polymerase chain reaction ; RT = room temperature ; T4MBP = Top 4 metal binding protein ; ICP-OES = inductively coupled plasma optical emission spectrometry ; ICP-MS = inductively coupled plasma mass spectrometry ; MS = mass spectrometry
date |
coworkers |
lab |
activity |
short summary |
08-Oct-2014 | Alina, Lisa | Inorganic chemistry | ICP-OES analysis | sample preparation with high pressure, heat and 2.5 % HNO3 / half-quantitative analysis: using calibration curves with defined heavy metal concentrations but only estimated sample volumina / detection via ICP-OES |
07-Oct-2014 | Anke | Botany | selection of transformed A. thaliana | potting transformed plants (without chlorosis) into substrat |
06-Oct-2014 | Alina, Andreas | IPG | E. coli preparation for MS analysis | E. coli Origami 2 cultures were precipitated by centrifuging them for 15 min at 4500 x g and 4 °C/ precipitates were dissolved in isoosmotic buffer/ after five washing steps with the same buffer, the precipitates were dried for 48 h at 70 °C |
03-Oct-2014 | Fabian, Katharina, Björn | IPG | preparation of new large scale E. coli cultures (zn, cu, cd) for MS | preparation of two repetitions of 500 ml: Origami 2_pASK with and without T4MBP and 0.25 mM cadmium, Origami 2_pASK with and without T4MBP and copper, Origami 2_pASK with and without T4MBP and zinc, Origami 2_pASK with and without T4MBP without heavy metals are used as controls/ all in all 16 flasks with 500 ml cultures/ induction of proteinexpression with anhydrotetracycline/ growing at 20 °C for 4 d to reach a high OD |
02-Oct-2014 | Fabian, Melanie | IPG | analysis of lethal concentration of copper and zinc | usage of copper-nitrate and zinc-nitrate/ analyses of growth rates of E. coli Origami 2 within media containing different heavy metal concentrations |
25-Sept-2014 | Fabian | Inorganic chemistry | ICP-MS analysis | sample preparation with high pressure, heat and 2.5 % HNO 3 / quantitative analysis: using calibration curves with defined heavy metal concentrations/ detection via ICP-MS |
22-Sept-2014 | Björn | IPG | pellitizing the E. coli cultures for MS | pellitizing the 2 l Origami 2 with pASK_T4MBP and 0.25 mM cadmium etc./ five washing steps with isoosmotic buffer/ drying the three pellets for 24 h and notation of dry weight |
19-Sept-2014 | Fabian | IPG | preparation of large scale E. coli cultures for MS | preparation of 2 l Origami 2 with pASK_T4MBP and 0.25 mM cadmium/ Chassis Origami 2 with and without cadmium (2 l each) are used as controls/ induction of proteinexpression with anhydrotetracycline/ growing at 25 °C for 3 d to reach a high OD |
19-Sept-2014 | Fabian | IPG | transformation of pASK (no insert) into Origami 2 | analyses: a comparable expression system for pASK_T4MBP is needed/ chemical competent cells were made/ transformation via heat shock, incubation over weekend at 16 °C |
19-Sept-2014 | Steffen | IPG | plasmidpreparation/ sequencing | plasmidpreparation of ONC (red/white colony pSB1C3) and colony T4MBP/ sequencing with primer 16; T4MBP sequenced with primer 16 and 17 |
18-Sept-2014 | Fabian | IPG | physiological test of T4MBP activity | Origami 2 with pASK_T4MBP at different cadmium concentrations (0 - 1 mM) were analyzed/ at 0.2 mM high grow rates were observed/ in comparison to Origami 2 without T4MBP no significant effect of TMBP was seen/ problem: induction of proteinexpression reduces growth rates: another inducible protein in Origami 2 is needed for comparison |
18-Sept-2014 | Steffen | IPG | colony-PCR | colony-PCR using E. coli colonies and primer 16 and 17: detection of 1 positive T4MBP-clone/ growth of red and white colonies (pSB1C3): colony-PCR |
17-Sept-2014 | Fabian, Steffen | IPG | immunostain | immunostaining blotted PVDF-membranes: use of Strep-tag- and His-tag-antibody (positive control): Strep shows specific signal at 37 kD, His shows signal at 37 kD as well/ Anti-Strep obviously works/ protein is found in inclusion bodies and supernatant |
16-Sept-2014 | Fabian, Steffen | IPG | SDS-PAGE/ Western Blot | testing of new Strep-tag-antibody/ use of pASK_T4MBP in Origami 2 to produce protein/ harvesting via ultrasound sonification/ test of protein pellet and supernatant/ run of discontinuous SDS-PAGE/ blotting proteins on PVDF/ transfer-check via Ponceau-stain/ membrane-blocking over night with Roti-Block |
16-Sept-2014 | Steffen | IPG | saving linearized pSB1C3 into E. coli / transformation of E. coli | ligation reactions with/without ligase: ligation for 1h at RT/ transformation of XL1-Blue Competent Cells via heat-shock using 10 µl ligation reaction/ selection on chloramphenicol/ transformation of XL1-Blue Competent Cells via heat-shock using 5 µl ligation reaction (11-Sept-2014)/ selection on chloramphenicol |
15-Sept-2014 | Fabian | Botany | selection of transformed A. thaliana seeds | sterilization of harvested A. thaliana seeds (09-Sept-2014) with ethanol/ plating of seeds on MSO-media with 2 % sucrose and 15 µg/ml phosphinothricin for selection/ stored for 2 days at 4 °C/ put at 20 °C until germination |
12-Sept-2014 | Steffen | IPG | colony-PCR/ sequencing results | colony-PCR using E. coli colonies (11-Sept-2014) and primer 16 and 17: 0 positive clone Expansin/T4MBP, results of expansin and CBD are positive |
11-Sept-2014 | Steffen, Anke | IPG | growth curves of Origami 2 pASK/ cloning T4MBP/ CBD into pSB1C3 (shipping vector)/ plasmidpreparation/ sequencing | growth of Origami 2 pASK in media containing five different concentrations of cadmium/ measurement of OD 600 over a period of 7 hours (1 per h)/ purification of PCR-products (10-Sept-2014)/ restriction digest of PCR-product and pSB1C3 with FastDigest EcoRI and PstI/ purification of digested products/ ligation for 1 h at RT/ transformation of XL1-Blue Competent Cells via heat-shock using 5 µl ligation reaction/ selection on chloramphenicol/ plasmidprep (glystocks prepared before) of ONC of positive colonies Expansin/CBD: sequencing with primer 16 |
10-Sept-2014 | Steffen | IPG | colony-PCR | colony-PCR using E. coli colonies (09-Sept-2014) and primer 16 and 17: 1 positive Expansin-clone/ ONC of positive colonies CBD/Expansin |
09-Sept-2014 | Steffen | IPG | colony-PCR | colony-PCR using E. coli colonies (08-Sept-2014) and primer 16 and 17: 1 positive CBD-clone/ transformation of XL1-Blue Competent Cells via heat-shock using 5 µl ligation reaction (05-Sept-2014)/ selection on chloramphenicol |
08-Sept-2014 | Fabian, Steffen | Botany | harvesting of transgenic seeds from A. thaliana | harvesting of mature seeds of transformed plants (transformation date: 31-July-2014) |
08-Sept-2014 | Steffen | IPG | transformation of E.coli XL1-blue with pSB1C3 | transformation of XL1-Blue Competent Cells via heat-shock using 5 µl ligation reaction (05-Sept-2014)/ selection on chloramphenicol |
05-Sept-2014 | Steffen | IPG | cloning T4MBP, Expansin, CBD into pSB1C3 (shipping vector) | test of FastDigest enzymes: expansin digested/not digested on 2.5% gel: digestion was positive/ purification of PCR-product (04-Sept-2014)/ restriction digest of PCR-product and pSB1C3 with FastDigest EcoRI and PstI/ purification of digested products/ ligation for 1 h at RT |
03-Sept-2014 | Steffen | IPG | colony-PCR | colony-PCR using E. coli colonies (02-Sept-2014) and primer 16 and 17: 0 positive clones |
02-Sept-2014 | Steffen | IPG | cloning T4MBP, Expansin, CBD into pSB1C3 (shipping vector) | purification of PCR-product (01-Sept-2014)/ restriction digest of PCR-product and pSB1C3 with NEB EcoRI and PstI/ purification of digested products/ ligation for 1 h at RT/ transformation of XL1-Blue Competent Cells via heat-shock/ selection on chloramphenicol |
20-Aug-2014 | Katharina, Björn | IPG | insertion of T4MBP in pASK | sequencing confirms the insertion of T4MBP in pASK |
19-Aug-2014 | Melanie | IPG | sequencing.v2 (same primer) | sequencing with primer 1261 (IPG) again stopped 10 bp before BglII-site: no definite result: again |
18-Aug-2014 | Katharina, Björn | IPG | insertion of T4MBP in pASK | sequencing of pASK with T4MBP not long enough |
14-Aug-2014 | Katharina | IPG | insertion of T4MBP in pASK | plasmid isolation: shipping to Seqlab |
13-Aug-2014 | Katharina | IPG | insertion of T4MBP in pASK | colony-PCR with primers 729 and 734: 1 positive colony: ONC |
12-Aug-2014 | Katharina, Björn | IPG | insertion of T4MBP in pASK | preparation of XL1-Blue Competent Cells/ transformation of XL1-Blue Competent Cells with ligation mixture |
11-Aug-2014 | Katharina | IPG | insertion of T4MBP in pASK | amplification of T4MBP with primers 8 and 9/ addition of EcoRI und NcoI sites via primer/ purification of PCR reaction mixture via kit/ digestion of pASK and amplificate with EcoRI and NcoI/ ligation over night (16 h, 16°C) |
11-Aug-2014 | Katharina, Björn | IPG | insertion of T4MBP in pASK | amplification of T4MBP by adding EcoRI and NcoI sites with the primers 8 and 9/ digestion of amplificate and pASK vector with EcoRI and NcoI/ simultaneous dephosphorylation of pASK/ ligation of pASK and T4MBP (16 h, 16 °C) |
10-Aug-2014 | Andreas | IPG | immunostain.v2 | still no difference between control and samples/ 9 min are enough for the final incubation of substrate buffer |
09-Aug-2014 | Andreas | IPG | colony-PCR/ SDS-PAGE.v2 | colony-PCR with primer 729 and 734 (IPG),T A = 48 °C (1:30 min) |
08-Aug-2014 | Andreas | IPG | pASK-transformation.v2 | transformation of freshly prepared heatshock competent BL21 (DE3) pLyss cells with Katharina's and Björn's ligation-product |
08-Aug-2014 | Katharina, Björn | IPG | insertion of T4MBP in pASK | isolation of pASK from ONC |
07-Aug-2014 | Andreas | IPG | immunostain/ ONC | polyclonal anti-flag-antibody (primary antibody) and anti-rabbit alkaline phosphatase (secondary antibody); both 1:2000 diluted/ after incubation with substrate buffer for 13 min: same signals (all over the lanes, very unspecific) in samples and control: maybe problems in sample handling: repetition/ ONC of pORE-E3 |
07-Aug-2014 | Katharina, Björn | IPG | insertion of T4MBP in pASK | only colonies on positive control: again inoculation of ONC |
06-Aug-2014 | Anke, Andreas | IPG | SDS-PAGE/ Coomassie-stain/ blotting | volume of cellulose-bound protein samples were reduced by direct application of „Polyethylenglykol 6000“ on top of the tube: all samples loaded into a 12 % SDS-PAGE |
06-Aug-2014 | Katharina, Björn | IPG | insertion of T4MBP in pASK | amplification of CDS (without His-tag) with primer 8 and 9 (Botany) via Phusion/ purification of the product with „Wizard-PCR and Gel Kit“/ double digestion of PCR product and pASK vector with EcoRI and NcoI for 30 min/ purification of both samples: poor results/ ligation of insert and pASK for 2 h (22 °C)/ transformation over night (37 °C) |
05-Aug-2014 | Andreas | IPG | cellulose-bound-protein GFP_in_pMA_EMP2 | dialysis (four times) to remove urea from the cellulose samples (1x overnight, 2x during the day, 1x overnight) |
05-Aug-2014 | Katharina, Björn | IPG | insertion of T4MBP in pASK | isolation (pORE_E3_2x35S_Expa_T4MBP_CBD) from E. coli ONC via plasmidpreparation (MiniKit)/ amplification of CDS (without His-tag) with primer 8 and 9 (Botany) via Phusion: did not work: again |
04-Aug-2014 | Anke, Andreas | IPG | isolation of transient expressed protein from N. tabacum | isolation of cellulose-bound and unbound protein from N. tabaccum : cooling via liquid nitrogen, automatically maceration by „Precellys“ and resuspendation in 1 x SDS-sample buffer (one sample for each plant)/ debris-pellet from centrifugation washed with dd H 2 0 two times/ incubation in 1 ml 8 M urea overnight |
31-July-2014 | Steffen, Fabian | Botany | floral dip transformation of A. thaliana | transformation of A. thaliana via floral dip by using A. tumefaciens as vector: used construct: pORE_E3_2x35S_Expa_T4MBP_CBD |
31-July-2014 | Anke, Björn | IPG | transient transformation of N. tabacum with pORE_E3_2x35S_Expa_T4MBP_CBD | transient transformation of 5 N. tabacum plants: used construct: pORE_E3_2x35S_TMBP in GV1301 and 4 plants without GV1301/ use of 1ml per leave and two leaves per plant |
21-July-2014 | Fabian | Botany | colony-PCR of Agrobacterium -transformation | colony-PCR with primer 11 (Botany) and 1484: poor results: proabably too high annealing temperature/ new colony-PCR with primer 11 and 1261/ preparation of 2 day cultures for Arabidopsis transformation |
18-July-2014 | Fabian | Botany | transformation of Agrobacterium with pORE_E3_2x35S_Expa_T4MBP_CBD | using electrocompetent GV3101 cells |
17-July-2014 | Steffen, Fabian | Botany | plasmidpreparation/ sequencing | use of ONC of E. coli with pORE and insert/ plasmidpreparation via Thermo Kit/ sequencing from both directions with primer 11 (Botany) and 1261: poor results for reverse direction/ resequencing of reverse sequence with primer 1484 |
15-July-2014 | Steffen, Fabian | Botany | colony-PCR of pORE with insert | Taq-PCR of 27 colonies using primer 11 (Botany, binds 35S) and 1012: poor results: wrong reverse primer used/ new colony-PCR with other reverse primer 1261 (binds NOS-terminator): result fine/ ONC |
14-July-2014 | Steffen, Fabian | Botany | cloning of CDS (Expa_T4MBP_CBD) into pORE2x35S | purification of Phusion-PCR (11-July-2014) to get rid of disturbing enzymes/ double digest of purified PCR-product and vector with MluI and BamHI for 1 h/ preparative gelelectrophoresis: cutting of specific bands/ gelextraction/ ligation of digested insert and vector for 1 h/ transformation of chemical competent E. coli DH5alpha with ligated DNA via heatshock |
11-July-2014 | Fabian | Botany | amplification of CDS from polyprotein | Phusion-PCR: using Geneart-shipping vector with insert as template, primer 106 and 859 |
03-July-2014 | Katharina | IPG | negative colony-PCR | continued by Fabian and Steffen |
02-July-2014 | Katharina | IPG | repetition of 27-June-2014: transformation of XL1–blue Competent Cells with T4MBP in pORE-E3 with 35S-Promotor.v2 | |
01-July-2014 | Katharina | IPG | ligation of T4MBP with pORE-E3 with 35S-Promotor.v2 | digestion of pASK with T4MBP and pORE-E3 with 2x35S promotor with MluI and BamHI/ overnight ligation |
30-June-2014 | Andreas | IPG | sequencing Bielefelder-CBDs | sequencing (Seqlab/ Microsynth) with primer 611 (GCTGGCCTTTTGCTCAGATGTTCTTTCCTGCGTTATC): optained sequencing result matches the (online) given sequence |
30-June-2014 | Katharina | IPG | negative colony-PCR | |
27-June-2014 | Katharina | IPG | transformation of XL1-Blue with T4MBP in pORE-E3 with 35S-Promotor.v1 | |
26-June-2014 | Katharina | IPG | ligation of T4MBP with pORE-E3 with 35S-Promotor.v1 | digestion of pASK with T4MBP and pORE-E3 with 2x35S promotor with Mlu1 and BamHI/ overnight ligation |
26-June-2014 | Katharina | IPG | plasmidpreparation of pASK | digestion of pASK with T4MBP and pORE-E3 with 2x35S promotor with Mlu1 and BamHI/ overnight ligation |
25-June-2014 | Katharina | IPG | colony-PCR of transformated XL1blue with T4MBP in pASK | primer for colony PCR: T7 and BackSeq-pGII |
24-June-2014 | Katharina | IPG | back-up-transformation of synthesised T4MBP located in pASK | transformation of XL1-Blue Competent Cells |
24-June-2014 | Melanie | IPG | linearization of Bielefelder-CBDs | linearizing BBa_K863101 & BBa_K863111 |
24-June-2014 | Melanie, Andreas | IPG | isolation of Bielelfelder-CBDs | isolation of BBa_K863101 & BBa_K863111 with PeqGOLD Plasmid Miniprep Kit by PeqLab GmbH and selfmade solutions |
20-June-2014 | Andreas | IPG | ONC of Bielefelder-CBDs | culturing XL1 containg pSB1C3 plasmid with parts BBa_K863101 & BBa_K863111 in 10 ml LB medium and 10 µl chloramphenicol |
19-June-2014 | Fabian | Botany | plasmidpreparation.v2 | preparation of the 3 E. coli clones which were expected to have the right insert (using Thermo Mini-Prep Kit)/ sequencing of all 3 clones using Primer 1011 (IPG): fine result: all 3 clones have the insert at the right position |
18-June-2014 | Fabian | Botany | new colony-PCR | new colony-PCR to detect transformed E. coli clones using primers 1011 and 1012 (IPG): 3 positive clones which were used for ONC |
16-June-2014 | Fabian | Botany | plasmidpreparation.v1 |
preparation of 2 E. coli clones which were expected to have the right insert (using Thermo Mini-Prep Kit)/ sequencing of one clone using Primer 962 (IPG): poor results: probably Primer 962 isn't working (even without 2x35S insertion sequencing should have worked) |
13-June-2014 | Fabian | Botany | colony-PCR | colony-PCR for detecting transformed E. coli clones using amplification primer 1 and 2 AND 1011 (IPG): poor results were interpreted wrongly: false clones were used for ONC |
12-June-2014 | Fabian | Botany | digest/ ligation/ transformation of 2x35S into pORE | gelextraction of 2x35S (using Qiagen-Kit)/ digest of 2x35S and pORE with XhoI and BamHI (37 °C for 1 h)/ gelelectophoresis for separation of cutted pORE, using the large fragment (gelextraction)/ ligation of 2x35S and pORE (using T7-Ligase)/ transformation of chemical competent E. coli DH5alpha / plating on LB-plates with kanamycin |
10-June-2014 | Fabian | Botany | amplification of 2x35S Promotor | use of primer 1 and 2 to amplify 2x35S promotor from template DNA/ separation of fragments via gelelectophoresis/ preparation of fragments from the gel |
21-May-2014 | Fabian | Botany | BamHI, MluI-digest | analysis whether both enzymes (BamHI and MluI) cut: double digest of pORE E3 |
19-May-2014 | Anke | IPG | - | preparation of a glystock |
19-May-2014 | Steffen, Fabian | Botany | - | plasmid isolation of pORE-E3 |
16-May-2014 | Steffen, Andreas | IPG | „over-weekend“- culture pORE-E3 | culturing JM109 bacteria cells containing pORE-E3 (250 µM from Glystock) in 10 ml LB media including 10 µM kanamycin over weekend at 27 °C |