Cloning of Human ABCB11 Gene in E. coli required the removal of an Intragenic Pribnow-Schaller Box before it’s Insertion into Genomic Safe Harbor AAVS1 Site using CRISPR Cas9

BackgroundGenomic safe harbors are sites in the genome which are safe for gene insertion such that the inserted gene will function properly, and the disruption of the genomic location doesn’t cause any foreseeable risk to the host. The AAVS1 site is the site which is disrupted upon integration of Adeno Associated Virus (AAV) and is considered a ‘safe-harbor’ in human genome because about one third of humans are infected with AAV and so far there is no apodictic evidence that AAV is pathogenic or disruption of AAVS1 causes any disease in man. Therefore, we chose to target AAVS1 site for the insertion of ABCB11, a bile acid transporter which is defective in Progressive Familial Intra Hepatic Cholestasis Type-2 (PFIC-2), a lethal disease of children where cytotoxic bile salts accumulate inside hepatocytes killing them and eventually the patient.MethodsWe used CRISPR Cas9 a genome editing tool to insert ABCB11 gene at AAVS1 site in human cell-lines.ResultsWe found that human ABCB11 sequence has a “Pribnow- Schaller Box” which allows its expression in bacteria and expression of ABCB11 protein which is toxic to E. coli and the removal of the same was required for successful cloning. We inserted ABCB11 at AAVS1 site in HEK 293T using CRISPR-Cas9 tool. We also found that ABCB11 protein has similarity with E. coli Endotoxin (Lipid A) Transporter MsbA.ConclusionWe inserted ABCB11 at AAVS1 site using CRISPR-Cas9, however, the frequency of homologous recombination was very low for this approach to be successful in-vivo (Figure: pictorial abstract).Pictorial AbstractABCB11 gene (which codes the transporter of human bile salts) is targeted to AAVS1 site using a construct which has 5’ and 3’ overhangs which are homologous to the AAVS1 site. A Pribnow box was detected inside ABCB11 gene which allowed the gene to transcribe in E. Coli causing bacterial lysis probably through competitive replacement of a homologous transporter protein in E. Coli (E. coli Endotoxin (Lipid A) Transporter) MsbA, resulting in Lipid A (L) accumulation inside the bacteria.

Structural and Biophysical Studies on Two Promoter Recognition Domains of the Extra-cytoplasmic Function σ Factor σC from Mycobacterium tuberculosis

σ factors are transcriptional regulatory proteins that bind to the RNA polymerase and dictate gene expression. The extracytoplasmic function (ECF) σ factors govern the environment dependent regulation of transcription. ECF σ factors have two domains σ2 and σ4 that recognize the -10 and -35 promoter elements. However, unlike the primary σ factor σA, the ECF σ factors lack σ3, a region that helps in the recognition of the extended -10 element and σ1.1, a domain involved in the autoinhibition of σA in the absence of core RNA polymerase. Mycobacterium tuberculosis σC is an ECF σ factor that is essential for the pathogenesis and virulence of M. tuberculosis in the mouse and guinea pig models of infection. However, unlike other ECF σ factors, σC does not appear to have a regulatory anti-σ factor located in the same operon. We also note that M. tuberculosis σC differs from the canonical ECF σ factors as it has an N-terminal domain comprising of 126 amino acids that precedes the σC2 and σC4 domains. In an effort to understand the regulatory mechanism of this protein, the crystal structures of the σC2 and σC4 domains of σC were determined. These promoter recognition domains are structurally similar to the corresponding domains of σA despite the low sequence similarity. Fluorescence experiments using the intrinsic tryptophan residues of σC2 as well as surface plasmon resonance measurements reveal that the σC2 and σC4 domains interact with each other. Mutational analysis suggests that the Pribnow box-binding region of σC2 is involved in this interdomain interaction. Interaction between the promoter recognition domains in M. tuberculosis σC are thus likely to regulate the activity of this protein even in the absence of an anti-σ factor.

Accessory Gene Regulator Control of Staphyloccoccal Enterotoxin D Gene Expression

ABSTRACT The quorum-sensing system of Staphylococcus aureus, the accessory gene regulator (Agr) system, is responsible for increased transcription of certain exoprotein genes and decreased transcription of certain cell wall-associated proteins during the postexponential phase of growth. This regulation is important for virulence, as evidenced by a reduction in virulence associated with a loss of the Agr system. The enterotoxin D (sed) determinant is upregulated by the Agr system. To define the Agr-regulated cis element(s) within the sed promoter region, we utilized promoters not regulated by Agr to create hybrid promoters. Hybrid promoters were created by using sed sequences combined with the enterotoxin A (sea) promoter or the S. aureus lac operon promoter sequences. The results obtained indicated that the Agr control element of the sed promoter resides within the −35 promoter element and at the Pribnow box to the +1 site of the promoter. At these positions of the sed promoter, a directly repeated 6-bp sequence was found. This repeat is important for overall promoter activity, and maximal regulation of the promoter activity requires both repeat elements. Furthermore, Agr control of sed promoter activity was found to be dependent upon the presence of a functional Rot protein. Therefore, the postexponential increase in sed transcription results from the Agr-mediated reduction in Rot activity rather than as a direct effect of the Agr system.

Rhodobacter capsulatus nifA1 Promoter: High-GC −10 Regions in High-GC Bacteria and the Basis for Their Transcription

ABSTRACT It was previously shown that the Rhodobacter capsulatus NtrC enhancer-binding protein activates the R. capsulatus housekeeping RNA polymerase but not the Escherichia coli RNA polymerase at the nifA1 promoter. We have tested the hypothesis that this activity is due to the high G+C content of the −10 sequence. A comparative analysis of R. capsulatus and other α-proteobacterial promoters with known transcription start sites suggests that the G+C content of the −10 region is higher than that for E. coli. Both in vivo and in vitro results obtained with nifA1 promoters with −10 and/or −35 variations are reported here. A major conclusion of this study is that α-proteobacteria have evolved a promiscuous sigma factor and core RNA polymerase that can transcribe promoters with high-GC −10 regions in addition to the classic E. coli Pribnow box. To facilitate studies of R. capsulatus transcription, we cloned and overexpressed all of the RNA polymerase subunits in E. coli, and these were reconstituted in vitro to form an active, recombinant R. capsulatus RNA polymerase with properties mimicking those of the natural polymerase. Thus, no additional factors from R. capsulatus are necessary for the recognition of high-GC promoters or for activation by R. capsulatus NtrC. The addition of R. capsulatus σ70 to the E. coli core RNA polymerase or the use of −10 promoter mutants did not facilitate R. capsulatus NtrC activation of the nifA1 promoter by the E. coli RNA polymerase. Thus, an additional barrier to activation by R. capsulatus NtrC exists, probably a lack of the proper R. capsulatus NtrC-E. coli RNA polymerase (protein-protein) interaction(s).

−11 Mutation in the ampC Promoter Increasing Resistance to β-Lactams in a Clinical Escherichia coli Strain

ABSTRACT A mutation was discovered in the Pribnow box of the ampC promoter in a clinical Escherichia coli strain. This −11 C-to-T transition created a perfect homology with the −10 consensus sequence. The new promoter was cloned upstream of the cat gene of pKK232-8 and induced a sixfold increase in promoter strength.