Transformation efficiencies

The bacterial one and two-hybrid systems have potential advantages over the yeast two-hybrid system due to the higher transformation efficiency and faster growth rate of coli. To date, however, the bacterial two-hybrid system has not been used for genome-scale analysis of protein-protein interactions. 

Table 2.1 Transformation efficiency, presented as the number of colon ies//rg plasmid DNA or as a percentage of the positive transformants of all cells transformed/transfected...

Some disadvantages are common for most eukaryotic expression systems lower growth rates, lower transformation efficiency, and often more expensive cultivation media compared with bacterial systems. 

A relatively low transformation efficiency of 103-105 colonies//rg DNA [85] and multicopy integration of expression cassettes make enzyme engineering and screening difficult. However, the first successful approaches using linear, integrative expression cassettes have been reported [86]. 

Campbell, E.I., Unkles, S.E., Macro, J.A. et al. (1989) Improved transformation efficiency of Aspergillus niger using the homologous niaD gene for nitrate reductase. Current Genetics, 16 (1), 53-56. 

In reactions closely related to the carbonylation processes described above, the dimeric azoarene palladium complexes (78) can be transformed efficiently in two steps into 3-imino-2-phenylindazolines (Scheme 95).162... 

Flanking counter-selectable markers Eliminates vector backbone Reduces transformation efficiency 23,24... 

A closely related dicationic platinum complex has been shown to transform efficiently /3-citronellene into cis-thujane in a highly diastereoselective manner, which mimics terpene biosynthesis.362 Also, using platinum(n) catalysis, Widenhoefer has reported an intramolecular alkylation of indoles with unactivated olefins, which can be carried out in an enantioselective fashion (Scheme 99).363... 

Catalytic olefin metathesis, in only a few years, has risen to be one of the most important and reliable processes in organic synthesis. Recently, several reports by Schrock and Hoveydallsbbond forming transformations efficiently and enan-tioselectively. A recent concise and enantioselective synthesis of exo-brevicomin by Burke utilizes chiral catalyst 91 (Scheme 13) to effect the desymmetriza-tion of 90 through a ring-closing metathesis.11531... 

An allenylaldehyde can be transformed efficiently into an a-methylene-y-butyro-lactone by a ruthenium-catalyzed carbonylative cycloaddition process (Scheme 16.34) [37]. The reaction mechanism may involve a metallacyclopentene, which undergoes insertion of CO and reductive elimination leading to the product. 

Interestingly, CNTs have been shown to be able to deliver exogenous genes not only in mammalian cells, but also in bacteria. Rojas-Chapana et al. (2005) demonstrated that oxidised, water-dispersible CNTs can deliver pDNA into E. coli (ratio of transformation efficiency/transformants of about 32) by opening up temporary nanochannels across the cell envelope. The authors described that addition of CNTs in a suspension containing E. coli and pDNA and application of a microwave frequency resulted in the orientation of the CNT tips perpendicularly to the cell surface and subsequently plasmid delivery into the bacteria. 

Both distillations must be performed with a pot temperature below 150°C In order to avoid thermal decomposition. NMR Indicates the product to be a 4 1 mixture of diastereomers. All available evidence denotes that both are transformed efficiently Into the cyclopentadlene. 

Dilute IMMEDIATELY with 40 p, TE Buffer (10 mM Tris, pH 8.0,1 mM EDTA) and either transform IMMEDIATELY or freeze the reaction until you are ready to transform. coli with a transformation efficiency in excess of 1 x 10 are recommended and if the vector has been modified for blue-white screening ensure that an appropriate E. coli host strain is used 5 pi of the diluted reaction should give tens to hundreds of colonies per well of a 24-well plate. 

Because JC8679 competent cells for electroporation are not commercially available, we had to produce our own. We routinely prepare competent cells following the protocol reported previously (14). Although JC8679 competent cells for electroporation of 10 -10 cfii/pg pUC19 are routinely prepared, the apparent transformation efficiency for the ORF trap is much lower (typically 50-100 colonies per 30 pL of transformation culture). 

Because a trap vector is prepared by PGR, a mutation within a vector region sometimes occurs. This is not a problem in most cases because we are interested only in an insert fragment, but in some cases, such a mutation happens to influence the vector fiinction (multimerization, reduction of transformation efficiency, etc.). This situation can be avoided by selecting another clone or transferring the insert to another intact vector. 

The two-hybrid yeast system has also been used as an approach to identifying host genes involved in transformation efficiency. As mentioned previously, VirE2 has been shown to interact with VIPl in Arabidopsis plants. Overexpression of VIPl in transgenic plants significantly increased the transformation efficiency as well as the rate of transformants that can accumulate. 

Dale, P. J., Hampson, K. K. (1995). An assessment of morphogenic and transformation efficiencies in a range of varieties of potato Solanum tuberosum L). Euphytica, 85, 101-108. 

Remove 1 - and 10-pL aliquots of the cells, and grow up on carbenicillin (50 pg/mL) LB plates in order to calculate the transformation efficiency/library size. 

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