TGATG vector: a new expression system for cloned foreign genes in Escherichia coli cells

Conditional Silencing of the <i>Escherichia coli pykF</i> Gene Results from Artificial Convergent Transcription Protected from Rho-Dependent Termination

“…All of the strains tested were derived from E. coli MG1655 as follows: (a) Red modifications of the chromosome were prepared according to Datsenko and Wanner [2000], exploiting the phage site-specific Xis/Int system instead of FLP recombinase, as described earlier ; (b) the cI ts857 carrier cassette from pPR53 [Mashko et al, 1990] ( fig. 4 a) was inserted into the native 80 attB site of the E. coli MG1655 genome according to Minaeva et al [2008] ( fig.…”

“…The ( cI ts857 -P R 1 cro -cat ) cassette, taken from plasmid pPR53 [Mashko et al, 1990], provided the ts-dependent biosynthesis of the hybrid Cro -CAT and the full- [Mashko et al, 1990] carrying the cassette with the phage cI ts857 gene and the gene for the hybrid Cro -CAT protein under the transcriptional control of the P R /O R regulatory region (P R ). CAT is chloramphenicol acetyltransferase originally encoding by the cat gene from pBR325 [Bolivar, 1978].…”

Direct Expression of Antimicrobial Peptides in an Intact Form by a Translationally Coupled Two-Cistron Expression System

“…To express a potent antimicrobial peptide BIIb (15) in its naturally occurring form, translationally coupled, two-cistron plasmids were constructed by aligning a coexpression partner gene as the first cistron and a BIIb gene as the second cistron. For the successful expression of large amounts of BIIb, the coexpression partner protein must be able to shield the host-lethal effects of BIIb by (i) forming a complex with the AMP upon expression (20) and (ii) reinforcing the formation of inclusion bodies to protect the expressed peptides from proteolytic degradation by host proteases. We chose hIFN-␥ as a coexpression partner because high-level expression of hIFN-␥ in E. coli results in the distribution of Ͼ90% of the accumulated gene product into inclusion bodies (32).…”

“…These changes lowered the isoelectric point (pI) of hIFN-␥ so as to efficiently facilitate complex formation (20) with cationic BIIb (the resulting modified hIFN-␥ was mIF). In addition, we introduced an SD sequence (termed SD2) into C terminus of the first cistron for the efficient translation of the gene product of the second cistron (Fig.…”

“…It has been reported that efficient translational coupling between two cistrons occurs when the cistrons are in close proximity to each other (13,20). Therefore, in order to analyze the effect of the distance between the coexpression partner and the AMP genes on the expression of AMPs, we made three different constructs (pmIFa-BIIb, pmIFb-BIIb, and pmIFcBIIb) in which we varied the distance between the termination codon of the first cistron (mIF) and the initiation codon of the second cistron (BIIb) (Fig.…”