 | | | | GPS™-M Mutagenesis System |
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Prices are in US dollars and valid only for US orders.
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Description:
The GPS™-M Mutagenesis System enables you to insert a transposable element of your own design into your target DNA by in vitro transposition. It uses TnsABC* Transposase to insert a Tn7-based transposon randomly into the DNA target (1,2,3). Target DNA may be a plasmid, cosmid, BAC or purified chromosomal DNA, depending on your interests (3-5). If the insertion site is within a translated gene segment, this will normally result in a null (loss of function) mutation. There is minimal site preference for insertion, so disruption of any open reading frame should be possible. Due to target immunity, only one insertion occurs per DNA molecule. Target immunity acts in vivo over a distance of ~190 kb (6,7). Therefore, the in vitro reaction produces a population of target DNA molecules each containing the transposable element at a different position.
To facilitate adaptation of the transposable element to your particular mutagenesis application, the transposon donor (pGPS3) can be customized by adding to or replacing the kanamycin resistance marker in the Transprimer (Figure 1). For ease of manipulation by the user, the donor plasmid can be grown easily in standard laboratory E. coli strains; the vector backbone carries Ampr and a pUC origin of replication. To destroy unreacted donor molecules and avoid undesirable reaction products, the donor can be destroyed by digestion with the rare-cutting enzyme PI-SceI (Figure 1). For applications in which the mutagenized DNA will be transformed into naturally-competent organisms (which take up single DNA strands), a protocol is supplied for filling-in and ligating the gaps left by the insertion transposition reaction.
Notes: The Kanr marker on the Transprimer is not designed to express in Gram positive organisms or eukaryotic cells. Transposon ends have stop codons in all frames, so translational fusions are not practical.
Figure 1: GPS-M Transposon donors.
Figure 2: Reaction Overview.
Advantages:- Transprimer insertion into the coding region of a gene will ordinarily produce a null mutation
- Transprimer is inserted randomly into the target DNA. There are no "hot spots" for insertion (3)
- Transprimer donor carries kanamycin resistance for easy detection
- Standard subcloning sites (BamHI, EcoRV) flank the Kanr marker so that this fragment can be replaced by the marker of your choice (Figure 2)
- Unique rare-cutting restriction enzyme sites (NotI, PmeI, I-CeuI, SwaI, SpeI) in the nonessential region of the Transprimer facilitate manipulation and insert mapping
- pGPS3 and customized donors can be grown in ordinary lab strains of E. coli
- The entire protocol takes less than 3 hours
- Sequencing primer binding sites in the Transprimer ends enable rapid location of the exact position of your insertion mutation
Kit Components:
BSA
Control Target Plasmid (LITMUS 28)
pGPS3 Transprimer Donor
PI-SceI
PrimerN (30-mer)
PrimerS (30-mer)
Start Solution (20X)
TnsABC* Transposase
GPS™ Buffer Pack (10X)
NEBuffer PI-SceI (10X)
Storage Conditions
Storage Temperature:
-20°C
References
- Craig, N.L. (1996) Curr. Top. Microbiol. Immunol., 204, 27-48.
- Stellwagen, A.E. and Craig, N.L. (1997) Genetics, 145, 573-585.
- Biery, M.C., Stewart, F.J., Stellwagen, A.E., Raleigh, E.A. and Craig, N.L. (2000) Nucl. Acids Res., 28, 1067-1077.
- Gwinn, M.L., Stellwagen, A.E., Craig, N.L., Tomb, J.F. and Smith, H.O. (1997) J. Bacteriol., 179, 7315-7320.
- Xu, Y., Murray, B.E. and Weinstock, G.M. (1998) Infect. Immun., 66, 4313-4323.
- Stellwagen, A.E. and Craig, N.L. (1997) EMBO J., 16, 6823-6834.
- DeBoy, R.T. and Craig, N.L. (1996) J. Bacteriol., 178, 6184-6191.
Reagents Sold Separately
BSA
pGPS3 Transprimer Donor
PI-SceI
PrimerN (30-mer)
PrimerS (30-mer)
TnsABC* Transposase
GPS™ Buffer Pack
NEBuffer PI-SceI
Companion Products
1 kb DNA Ladder
GPS™-1 Genome Priming System
GPS™-LS Linker Scanning System
I-CeuI
I-SceI
NotI
PmeI
SpeI
SwaI
Legal
Patents:
Johns Hopkins University: Licensed Under U.S. Patent No. 6,420,524
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