Vectors
Below are maps and sequences for some of the plasmids for stable transformation of Trypanosoma spp. available from the Wickstead lab or collaborators.
In addition to images, there are annotated GenBank format files containing both sequence and features. These are designed to be used with M. Wayne Davis's ApE: A Plasmid Editor, which is free, Open Source and runs on Windows/MacOS-X/Linux, but should also be compatible with your lab's preferred vector analysis software. Still included for some of the older plasmids are annotated Gene Construction Kit files, but we are no longer using this proprietary format.
A small perl script that we use to convert annotated ApE/GenBank files and make a pretty SVG files from them (avaiable here). The maps below were generated with this script.
Plasmids for genetic modification of T. congolense
The following are the base vectors generated in
Awuah-Mensah et al. (2021) PLoS Path 17:e1009224 as species-specific constructs for reliable, scalable functional genetics in bloodstream-form
Trypanosoma congolense.
| Plasmid |
map |
GenBank |
Integration site |
Promoters |
Transgenes |
Marker |
| pTcoSM |
 |
 |
TUBB |
n/a |
T7RNAP, TetR |
PAC |
| pGad10-TcoTUB |
|
|
TUBB |
n/a |
GFP |
HYG |
| pTcoLUC1 |
|
|
P(rRNA) |
RRNA |
LUC |
NEO |
| pTcoLUC2 |
|
|
P(rRNA) |
RRNA |
LUC |
NEO |
| pTcoSce-tiT7 |
|
|
MC-VSG |
T7+tiT7 |
SCE |
HYG |
| pTcoSce-TbrRNA |
|
|
MC-VSG |
T7+tiTbRRNA |
SCE |
HYG |
| p2T7-TcoV |
|
|
MC-VSG |
RRNA+2xtiT7 |
n/a |
NEO |
| p3T7-TcoV |
|
|
MC-VSG |
T7+2xtiT7 |
n/a |
NEO |
| p3T7-TcoV-TT |
|
|
MC-VSG |
T7+2xtiT7 |
n/a |
NEO |
(Zipped archives of complete sets can be downloaded here)
Plasmids for RNAi in T. brucei
Simple modifications to the popular p2T7 and
p2T7TiTA plasmids which result in integration to the minichromosomal repeats, providing a tighter off state for knockdown of sensitive transcripts. These plasmids are described in
Wickstead et al. (2002) MBP 125:211-6. My thanks to
David Wildridge at the University of Glasgow who put together the Vector NTI versions of the plasmids that appear below.
| Plasmid |
map |
GenBank |
GCK |
VectorNTI |
Marker |
| p2T7-177 |
|
|
 |
 |
BLE |
| p2T7-TA177 |
|
|
|
|
HYG |
(Zipped archives of complete sets can be downloaded here)
Plasmids for inducible transgene expression (pGad)
Constructs for inducible expression of transgenes from various genomic locations. The pGad series contain a single 2-gene polycistron (
GFP
and
Drug) under the influence of a tetracycline-inducible promoter (c.f. dual-promoter
pDex series). These vectors are described in
Wickstead et al. (2003) NAR 31:3993-4000.
| Plasmid |
map |
GenBank |
GCK |
Integration site |
Inducible promoter |
Transgene |
Marker |
| pGad7 |
|
|
na |
- |
EP1 |
GFP |
HYG |
| pGad8-rDNA |
|
|
|
rDNA spacer |
EP1 |
GFP |
HYG |
|
| pGad8-3x177 |
|
|
|
177bp repeat |
EP1 |
GFP |
HYG |
|
| pGad8-V4 |
|
|
|
VSG-G4 |
EP1 |
GFP |
HYG |
|
| pGad8-V8 |
|
|
|
VSG-S8 |
EP1 |
GFP |
HYG |
|
| pGad8-Tub |
|
|
|
TUB |
none |
GFP |
HYG |
|
| pGad9-rDNA |
|
|
|
rDNA spacer |
T7 |
GFP |
HYG |
|
| pGad9-177 |
|
|
|
177bp repeat |
T7 |
GFP |
HYG |
|
| pGad9-V4 |
|
|
|
VSG-G4 |
T7 |
GFP |
HYG |
|
(Zipped archives of complete sets can be downloaded here)
Plasmids for inducible transgene expression (pDex)
Minichromosomal repeat-integrating constructs for inducible expression of transgenes. pDex177, 277, 377 and 477 are derivatives of
pLEW100 with various promoters. pDex577 vectors are highly-modular expression vectors designed and constructed by
Steve Kelly. These vectors are described in
Kelly et al. (2007) MBP 154:103-9 along with a large number of modified forms of pDex377 produced in the lab of
Mark Carrington.
| Plasmid |
map |
GenBank |
GCK |
Constitutive promoter |
Marker |
Inducible promoter |
Transgene |
| pDex177 |
|
|
|
T7 |
BLE |
EP1 |
LUC |
|
| pDex277 |
|
|
|
T7 |
BLE |
T7 |
LUC |
|
| pDex377 |
|
|
|
rDNA |
HYG |
EP1 |
LUC |
|
| pDex477 |
|
|
|
rDNA |
BLE |
EP1 |
LUC |
|
| pDex477-Y2 |
|
|
na |
rDNA |
BLE |
EP1 |
TY-YFP-TY |
|
| pDex577-G |
|
|
|
rDNA |
BLE |
T7 |
TY-GFP-TY |
|
| pDex577-Y |
|
|
|
rDNA |
BLE |
T7 |
TY-YFP-TY |
|
| pDex577-C |
|
|
|
rDNA |
BLE |
T7 |
TY-GFP-TY |
|
(Zipped archives of complete sets can be downloaded here)
Plasmids for endogenous locus tagging: C-terminus
A set of plasmids designed for medium-throughput tagging of trypanosomal proteins at the C-terminus. The plasmids allow for easy incorporation of the endogenous locus 3'-intergenic DNA - to express chimeric proteins at levels similar to wild-type. Currently unpublished.
| Plasmid |
map |
GenBank |
GCK |
Marker |
Tag |
|
| pEnG0 |
|
|
|
HYG |
GFP |
|
| pEnG0B |
|
|
|
BLAST |
GFP |
|
| pEnG0P |
|
|
|
PURO |
GFP |
|
| pEnT0 |
|
|
|
HYG |
Myc-TY |
|
| pEnTT0 |
|
|
|
HYG |
2xTY |
|
| pEnTT0B |
|
|
|
BLAST |
2xTY |
|
| pEnTT0P |
|
|
|
PURO |
2xTY |
|
(Zipped archives of complete sets can be downloaded here)
Plasmids for endogenous locus tagging: N-terminus
A set of plasmids designed for tagging of trypanosomal proteins at the N-terminus. Similar in style to the C-terminus tagging constructs above. Currently unpublished.
| Plasmid |
map |
GenBank |
GCK |
Marker |
Tag |
|
| pEnNY0 |
|
|
|
HYG |
YFP |
|
| pEnNTYY0 |
|
|
|
HYG |
TY-YFP |
|
| pEnNmSt0-B |
|
|
n/a |
BLAST |
mStrawberry |
|
| pEnNmSt0-N |
|
|
n/a |
NEO |
mStrawberry |
|
(Zipped archives of complete sets can be downloaded here)
Plasmids for endogenous locus tagging: either terminus
This set of plasmids were designed and constructed by
Steve Kelly as a set of highly modular constructs for easy tagging of endogenous genes at either N- or C-termini. They are described in
Kelly et al. (2007) MBP 154:103-9 and are provided here as a service.
| Plasmid |
map |
GenBank |
GCK |
Marker |
Tag |
|
| pEnT5-G |
|
|
|
HYG |
TY-GFP-TY |
|
| pEnT5-Y |
|
|
|
HYG |
TY-YFP-TY |
|
| pEnT5-C |
|
|
|
HYG |
TY-CFP-TY |
|
| pEnT6B-G |
|
|
|
BLAST |
TY-GFP-TY |
|
| pEnT6B-Y |
|
|
|
BLAST |
TY-YFP-TY |
|
| pEnT6B-C |
|
|
|
BLAST |
TY-CFP-TY |
|
| pEnT6P-G |
|
|
|
PURO |
TY-GFP-TY |
|
| pEnT6P-Y |
|
|
|
PURO |
TY-YFP-TY |
|
| pEnT6P-C |
|
|
|
PURO |
TY-CFP-TY |
|
(Zipped archives of complete sets can be downloaded here)
