StarGate® Transfer Cloning Principle in Detail

Gene transfer into Acceptor Vector

In the transfer reaction step, the GOI may be transferred from the Donor Vector into a variety of Acceptor Vectors providing the desired genetic surroundings (i.e. tag, promoter (Prom), signal sequence etc.) by means of StarCombinase2. The resulting Destination Vector places the GOI under control of the selected promoter (prom) allowing GOI expression in the selected expression host. In addition, a tag (green) is fused to the C-terminal end of the GOI expression product in this example.

Efficient Subcloning using the StarGate® System

Using StarCombinaseTM the gene for GFP was transferred from a Donor Vector into a pPSG-IBA Acceptor Vector. After an 1 hour incubation, E. coli was transformed with the reaction mixture. All resulting clones exhibited green fluorescence by GFP expression. This provides evidence that the StarGate® subcloning reaction had performed accurately and reliably to put the GFP gene under control of the T7 promoter.

Reaction Efficiency >99%

Transformation of E. coli with a StarGate® transfer reaction mixture typically leads to more than thousand white colonies harbouring the desired Destination Vector. The presence of a few blue colonies only indicates a reaction efficiency far beyond 99 %.

Transfer Efficiency is Independent of Gene Size

The green fluorescent protein (GFP, 700 bp), bacterial alkaline phosphatase (BAP, 1400 bp) and T7 RNA polymerase (T7RNAP, 2650 bp) were transferred from the respective Donor Vector into all available Acceptor Vectors (cf. Table, page 4). The result provides evidence that the StarGate® subcloning procedure is almost not influenced by the length of the transferred gene and that even large genes can be transferred efficiently.

Transfer efficiency and fragment size

Colony numbers and fragment size

Technical information

E. coli is transformed with the mixture potentially including all 4 possible vectors. Due to selection on ampicillin plates, Donor Vector and by product – which provide a kanamycin resistance gene only – will not enable growth of E. coli. Acceptor Vector and Destination Vectors, however, enable growth due to the encoded ampicillin resistance genes. The Acceptor Vector carries the LacZa gene and, therefore, produces blue colonies on X-gal containing plates while LacZa has been replaced by GOI in the Destination Vector which, therefore, generates white colonies. The Destination Vector of this example puts GOI under control of the CMV promoter enabling GOI expression in mammalian cells and fuses a tag to the C-terminal end of GOI expression product.

References

Belfield, EJ, Hughes RK, Tsesmetzis, N, Naldrett, MJ, Casey, R (2007).
The gateway pDEST17 expression vector encodes a –1 ribosomal frameshifting sequence.
NAR 35 (4), 1322-1332.
Boshart, M, Weber, F, Jahn, G, Dorsch-Häsler, K, Fleckenstein, B, and Schaffner, W (1985).
A Very Strong Enhancer is Located Upstream of an Immediate Early Gene of Human Cytomegalovirus.
Cell 41, 521-530.
Gietz, RD, Sugino, A (1988).
New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene 74, 527-34. Macreadie, IG, Horaitis, O, Verkuylen, AJ, Savin, KW (1991). Improved shuttle vectors for cloning and high-level Cu⁽²⁺⁾-mediated expression of foreign genes in yeast
Gene 104, 107-11.
Nelson, JA, Reynolds-Kohler, C, and Smith, BA (1987).
Negative and Positive Regulation by a Short Segment in the 5´-Flanking Region of the Human Cytomegalovirus Major Immediate-Early Gene.
Mol. Cell. Biol. 7, 4125-4129.
Skerra, A. (1994).
Use of the tetracycline promoter for the tightly regulated production of a murine antibody fragment in Escherichia coli.
Gene 151, 131-135.
Studier, FW, Rosenberg, AH, Dunn, JJ, Dubendorff, JW (1990).
Use of the T7 RNA polymerase to direct expression of cloned genes.
Meth. Enzymol. 185, 60-89.

사용 후기: 사용후기가 없습니다.

사용후기 작성