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From 2014.igem.org

Notebook / Locate GFP and the CBD

Here we list all the steps that were required to locate the cellulose binding domain (CBD) within the cell using GFP as a marker.

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 ; EMP = Exponential Megapriming PCR ; GFP = Green fluorescent protein

date	coworkers	lab	activity	short summary
09-Sept-2014	Fabian, Anke	Biophysics	confocal microscopy	microscopy of transformed B. sinuspersici and N. tabacum (04-Sept-2014)
04-Sept-2014	Fabian	Botany	transient plant transformation	A. tumefaciens mediated transformation of B. sinuspersici (vacuum ilfiltration) and N. tabacum (leaf infiltration): used constructs: pORE_Expa~GFP~CBD, pEarley103_GFP, pEarley103_Plasmamembranemarker
01-Sept-2014	Fabian, Steffen, Anke, Andreas	Biophysics	confocal microscopy	microscopy of transformed B. sinuspersici and N. tabacum (transformation date: 29-Aug-2014)
29-Aug-2014	Fabian	Botany	transient plant transformation	A. tumefaciens mediated transformation of B. sinuspersici (vacuum infiltration) and N. tabacum (leaf infiltration): used constructs: pORE_Expa~GFP~CBD, pEarley103_GFP, pEarley103_Plasmamembranemarker
22-Aug-2014	Fabian	Botany	colony-PCR	colony-PCR using E. coli colonies (20-Aug-2014) and primer 11 (Botany) and 1261: 15 positive clones/ ONC of 3 of 15 positives clones
21-Aug-2014	Fabian	Botany	cloning Expa~GFP~CBD into pORE	purification of PCR-product (19-Aug-2014)/ restriction digest of PCR-product and pORE E3 2x35S with BamHI and MluI/ gelextraction of digested vector and PCR-product/ ligation for 1 h/ transformation of XL1-Blue Competent Cells via heat-shock/ selection on kanamycin
19-Aug-2014	Fabian	Botany	Phusion PCR	Phusion PCR using Expa~GFP~CBD-sequence from EMP as template
08-Aug-2014	Andreas	IPG	plasmid-isolation	isolation of pORE-E3 for future cloning and EMP_in_pMA_Colony1 for sequencing
07-Aug-2014	Andreas	IPG	colony-PCR	although colony-PCR (F1+R1) confirmed insertion of GFP: another primer set (1101 and 625, IPG) targeting the backbone of pMA did not bind in the colony, but showed an unexpected amplificate length of ~1200 bp in the negative control (expected for pMA: ~1600 bp)
05-Aug-2014	Andreas	IPG	cellulose-bound-protein GFP_in_pMA_EMP2	repetition of EMP-PCR(2) reaction mix no. 2 (protocol of Anke): gel confirmed the correct fragment length/ purification using the „Wizard-Kit“/ phosphorylation/ ligation overnight according to Anke's protocol
25-July-2014	Anke	IPG	colony-PCR of EMP2-transformation.v2	colony-PCR with primer 1011 and 625 (IPG) and 10 new colonies: poor results
23-July-2014	Anke	IPG	colony-PCR of EMP2-transformation.v1	colony-PCR with primer 1011 and 625 (IPG): poor results
22-July-2014	Anke	IPG	transformation of E. coli with EMP2	transformation of XL1-Blue Competent Cells
21-July-2014	Anke, Andreas	IPG	EMP2 GFP	screening the functional amount of megaprimer with and without F1: 20 ng megaprimer + F1 + R2
18-July-2014	Anke	IPG	EMP1 GFP	EMP1 with F1, R2 and GFP-vector to generate megaprimer: fine result