![]() The HI-Control strains feature a specially engineered lacI allele that expresses ∼200-fold more lac repressor protein than native chromosomal lacI, providing enhanced control over the leaky expression common in other T7 systems. Confirmed clones constructed in HI-Control 10G cells are transferred to HI-Control BL21(DE3) cells for protein expression. The kits are provided with two types of competent cells: HI-Control ™ 10G cells for stable clone construction, and HI-Control BL21(DE3) cells for expression from the T7 promoter. The Expresso T7 kits include pETite ® vectors, containing the bacteriophage T7 promoter under the control of lac operators. The Expresso SUMO (small ubiquitin-like modifier) vectors allow fusion to an N-terminal, cleavable 6×His-SUMO tag for enhanced soluble expression of difficult target proteins. Currently available Expresso System vectors include sequences encoding either N- or C-terminal hexahistidine (6×His) tags, permitting convenient target-protein purification by immobilized metal-affinity chromatography (IMAC). Additional sequences, such as short fusion tags or protease cleavage sites, can be introduced via primer design as desired.Įxpresso kits are available with preprocessed vectors containing either of two inducible promoters (T7 or rhamnose), and in several different configurations for expression of target proteins with convenient fusion tags for enhanced expression and purification ( Fig. End points of the target protein can be selected at will. Seamless fusion to the vector eliminates undesirable amino acids encoded by restriction sites or site-specific recombination sites. For most genes, more than 90% of colonies will have the target gene correctly inserted into the vector.Įlimination of the requirement for PCR product cleanup and enzyme treatment not only saves the cost of enzymes and multiple incubation and sample-handling steps but also simplifies the design of expression clones. The design and preparation of the Expresso System vectors ensure minimal background transformation with non-recombinant clones. Recombination between the vector and PCR product within the host cells precisely fuses the target gene to the vector in the proper orientation. To clone by Expressioneering, an aliquot (typically 1 ml) of unpurified PCR product is mixed with the preprocessed Expresso vector and immediately transformed into the chemically competent cells provided. I assume, the second recipe is more suitable to be used as a metaphor for a programming language that has type system.Instant cloning with Expressioneering Technology (As computer scientists know, JavaScript is dynamically typed which may cause many undesirable side effects according to the type theory. ![]() However, as the author likes it that way, probably he uses such a metaphor in his book. People who are into coffee mostly prefer pure coffee. ![]() The first one is more sophisticated and uncommon with many side flavors in it. ![]() So, the second explanation given in the book is the standard way. Normally, it is prepared in a bit longer time than normal espresso. I don't know how to write in the pronounciation alphabet, but it is pronounced something similar to "a-lawn-jee".
0 Comments
Leave a Reply. |