Hard- and software 4.1.1.
This work was written on an Asus N56V (ASUSTeK COMPUTER INC., Taiwan, China) using Microsoft Word 2010 (Microsoft Corporation, Redmond, WA, USA). For the design of diagrams Sigma Plot 11.0 (Systat Software Inc., San José, CA, USA) was used.
Chemiluminescence signals were detected with the 'LAS-4000' system (Fujifilm Europe, Düsseldorf). Autoradiographies were digitized using the 'FLA-7000' phosphorimager (Fuji Film Europe, Düsseldorf). Absorbance measurements on well plates were done using 'FLUOstar Omega' (BMG Labtech, Ortenberg) according with its own control software and 'MARS data analysis' software. Images were processed with 'Adobe Photoshop CS4' (Adobe Systems Inc., San Jose, CA, USA). Figures were generated using Microsoft PowerPoint 2010 (Microsoft Corporation, Redmond, WA, USA). T. brucei DNA and protein sequences were received from www.genedb.org. Analysis of DNA sequences and virtual design of plasmids was done with 'DNASTAR Lasergene' (GATC Biotch, Konstanz). For sequence alignments a service of the European Bioinformatics Institute ('EMBOSS needle pairwise sequence alignment' algorithm, www.ebi.ac.uk) was used. Prediction of APC/C recognition motif was done with 'GPS-ARM 1.0' (the cuckoo workgroup, China, arm.biocuckoo.org). For analysis of protein domain architecture the European Molecular Biology Laboratory online tool 'SMART' (smart.embl-heidelberg.de) was used. The National Center for Biotechnology Information (www.ncbi.nhi.gov/) provided electronic online services for literature research.
Chemicals, reagents and kits 4.1.2.
All chemicals and reagents were purchased, unless noticed otherwise, from AppliChem (Darmstad), BioRad (Munich), Calbiochem (via Merck, Darmstadt), Fermentas (St. Leon-Rot), GE Healthcare (München), Merck (Darmstadt), Invitrogen (via Thermo Scientific, Schwerte), Macherey-Nagel (Düren), New England Biolabs (NEB, Frankfurt a. M.), Qiagen (Hilden), Roth (Karlsruhe), Serva (Heidelberg), Sigma-Aldrich (Steinheim) and Thermo Scientific (Schwerte).
For all solutions, de-ionized sterile water was used. If needed, solutions were sterilised and sterile flasks were used.
| 86 Antibodies
4.1.3.
Commercial and self-made antibodies were used in this study as follows (Table 5 and 6).
Table 5: Primary antibodies.
antibody class and species antigen dilution source/reference BiP mouse monoclonal BiP 1:3000 WB Gertrud Lallinger-Kube,
Molecular Parasitology, University of Bayreuth
GFP IgG
mouse monoclonal
GFP 1:1000 WB Markus Hermann, Genetics, University of Bayreuth
His IgG1
mouse monoclonal
Histidin 1:2000 WB Santa Cruz Biotechnology, Dallas, TX, USA
antibody antigen and species dilution source
Atto 488
DNA oligonucleotides for this study were received from Microsynth (Balgach, Switzerland).
Their design was as follows (Table 7, 8 and 9).
Material and methods
| 87 Table 7: DNA oligonucleotides for changing multiple cloning sites and tags in vectors.
oligonucleotide sequence 5'->3' purpose
Table 8: DNA oligonucleotides for TbKif13-1 constructs.
oligonucleotide sequence 5'->3' purpose
pCS2tag_Kif13_1f Fse 5'-ATA GGC CGG CCG GCG AAG TGG GAA TTA
AAG CTG-3'
TbKif13-1 N-terminus, without startcodon; starts at 4th base of
TbKif13-1, FseI; forward; pHD180TbKif13-1, pTrc C FA, pcDNA5, pcDNA3.1, pCS2-eGFP 13-1 NES_ATG_forFs 5'- ATA GGC CGG CCA
TGG AAG AGT TCG TAG startcodon; starts at 94th base of TbKif13-1; FseI; forward; pHD1801, pTrc startcodon; starts at 175th base of TbKif13-1; FseI; forward; pHD1801, pTrc startcodon; starts at 1396th base of
TbKif13-1; FseI; forward; pHD1801
| 88 Kif13.1MycEGFPrev 5'-GGC GCG CCC TAA
ATC CCG TTT TGC TCG AGA-3'
TbKif13-1 C-terminus, with stopcodon;
ends at 2073rd base of TbKif13-1; AscI;
reverse; pHD1801, pTrc C FA, pcDNA5, stopcodon; ends at 1392nd base of TbKif13-1; AscI; reverse; pHD1801, pTrc 2073th base of TbKif13-1; AscI; reverse;
pHD1801 13-1 N-term GFP_re 5'-ATA GGC GCG CCC
GTG ATA CAA ATT CAC Kif13.1 143SA for 5'-GGG AGG TTA AAC
GGC GTA AAG CCC GCA Kif13.1 143SA rev 5'-CCA CGA TGC GGG
CTT TAC GCC GTT TAA Kif13-1 G371A for 5'-CTT TTA TTG ATC
TCG CTG CGA GTG AGC Kif13-1 G371A rev 5'-CCC ACG CTC ACT
CGC AGC GAG ATC AAT Kif13-1 E373A for 5'-CGC TGG GAG TGC
GCG TGG GGC GG-3'
TbKif13-1 E373A mutation within one ATP binding motif in the motordomain;
in order to prevent ATPase activity and maintain depolymerisation activity;
forward; pTrc C FA Kif13-1 E373A rev 5'-CCG CCC CAC GCG
CAC TCC CAG CG-3'
TbKif13-1 E373A mutation within one ATP binding motif in the motordomain;
in order to prevent ATPase activity and maintain depolymerisation activity;
reverse; pTrc C FA
Material and methods
| 89 Table 9: DNA oligonucleotides for TbAuk1 constructs.
oligonucleotide sequence 5'->3' purpose
pET-Auk1-forFseI 5'-ATA GGC CGG CCG AGG TCA ACT GAG GTC
GGG CGT-3'
TbAuk1 N-terminus, without startcodon; starts at 4th base of TbAuk1;
FseI; forward; pHD1801
TbAukT184A for 5'-ACC GTC GCA AGG CAT CTT GCG GGA
The multiple cloning sites (MCS) of all used vectors were provided with unique FseI and AscI sites for easier and more rapid subcloning. Sequencing of PCR product inserts was done by Macrogen (Macrogen Europe, Amsterdam, the Netherlands) according to their instructions.
The following vectors and plasmids were used during this study (Table 10 and 11).
| 90 Table 10: Vectors.
vector tag description and origin
pTrc C FA N-His6 inducible expression vector in E. coli; trc promoter;
ampicillin resistance; Thermo Scientific (Schwerte);
modified by Bianca Kakoschky, Molecular Parasitology, University of Bayreuth pHD1800GFP-myc GFP-myc-C inducible expression vector in T. brucei; procyclin
promoter; ampicillin and hygromycin resistance;
origin pHD1700, Dr. Voncken, University of Hull;
modified in this study
pHD1800 myc-C inducible expression vector in T. brucei; procyclin promoter; ampicillin and hygromycin resistance;
origin pHD 1700, Dr. Voncken, University of Hull;
modified in this study
pHD1801 N-myc inducible expression vector in T. brucei; procyclin promoter; ampicillin and hygromycin resistance;
origin pHD1701, Dr. Voncken, University of Hull;
modified in this study
pCS2-eGFP N-eGFP constitutive expression vector in mammlian cells;
simian cytomegalovirus (CMV) IE94 enhancer/promoter; ampicillin and hygromycin resistance; Turner and Weintraub (1994); modified
by department of Genetics, University of Bayreuth pcDNATM
5/FRT/TO-eGFP-FA
N-eGFP inducible expression vector for the 'Flp-InTM T-RExTM System' (Invitrogen); hybrid human CMV/TetO2 enhancer/promoter; contains FLP recombination site
(FRT) for Flp recombinase-mediated integration into the genome; ampicillin and hygromycin resistance;
Invitrogen via Thermo Scientific (Schwerte); modified by department of Genetics, University of Bayreuth pcDNATM3.1-eGFP-FA N-eGFP inducible expression vector in mammalian cells,
hybrid human CMV/TetO2 enhancer/promoter, ampicillin and neomycin resistance; Invitrogen via
Thermo Scientific (Schwerte); modified by department of Genetics, University of Bayreuth and
in this study
Material and methods
| 91 Table 11: Plasmids.
plasmid insert tag vector origin
pTrc-TbKif13-1 FL TbKif13-1 FL N-His6 pTrc C FA this study pTrc-TbKif13-1 ½ N + C TbKif13-1 ½ N + C N-His6 pTrc C FA this study pTrc-TbKif13-1 NM + C TbKif13-1 NM + C N-His6 pTrc C FA this study pTrc-TbKif13-1 NM TbKif13-1 NM N-His6 pTrc C FA this study pTrc-TbKif13-1 N + M TbKif13-1 N + M N-His6 pTrc C FA this study pTrc-TbKif13-1 ½ N + M TbKif13-1 ½ N + M N-His6 pTrc C FA this study pTrc-TbKif13-1 N + D-box TbKif13-1 N +
D-box
N-His6 pTrc C FA this study pTrc-TbKif13-1 ½ N + D-box TbKif13-1 ½ N +
D-box
N-His6 pTrc C FA this study pTrc-TbKif13-1 FL G371A TbKif13-1 FL G371A N-His6 pTrc C FA this study pTrc-TbKif13-1 FL E373A TbKif13-1 FL E373A N-His6 pTrc C FA this study
pHD1800-GFPmyc GFP myc-C pHD1800 this study
pHD1800-TbKif13-1 NGFP-myc TbKif13-1 N GFP-myc-C
pHD1800GFP
-myc
this study pHD1800-TbKif13-1 ½NGFP-myc TbKif13-1 ½ N
GFP-myc-C
pHD1800GFP
-myc
this study pHD1801-TbKif13-1 FL TbKif13-1 FL N-myc pHD1801 this study pHD1801-TbKif13-1 ½ N + C TbKif13-1 ½ N + C N-myc pHD1801 this study pHD1801-TbKif13-1 NM + C TbKif13-1 NM + C N-myc pHD1801 this study pHD1801-TbKif13-1 NM TbKif13-1 NM N-myc pHD1801 this study pHD1801-TbKif13-1 N + M TbKif13-1 N + M N-myc pHD1801 this study pHD1801-TbKif13-1 ½ N + M TbKif13-1 ½ N + M N-myc pHD1801 this study pHD1801-TbKif13-1 C TbKif13-1 C N-myc pHD1801 this study
pHD1801-TbAuk1 TbAuk1 N-myc pHD1801 this study
pHD1801-TbAuk1 K58R TbAuk1 K58R N-myc pHD1801 this study pHD1801-TbAuk1 T184A TbAuk1 T184A N-myc pHD1801 this study pQE60-TbHistoneH3 TbHistoneH3 His6-C pQE60 Larry Ruben,
Southern Methodist University, Dallas, USA pCS2-eGFP-TbKif13-1 FL S143A TbKif13-1 FL S143A N-eGFP pCS2-eGFP this study
pcDNA5/FRT/TO-eGFPTbKif13-1
TbKif13-1 FL S143A N-eGFP pcDNATM3.1 -eGFP
this study
pAG1786 Flp-recominase - pCS2 Genetics,
University of Bayreuth
| 92 4.2. Microbiological techniques
E. coli strains and media