3 | P a g e 4 | P a g e Plasmid constructs.pCNR1prom-Luc. The human genomic fragment containing the CNR1 promoter (CNR1 prom) was amplified from human placental DNA using the following primers (9):CNR1prom for; 5'-GATAACCTTTTCTAACCACCCACCTAG-3', CNR1prom rev. 5'-GCGGAAAAGAAGTGGAGAAG-3' and cloned into the EcoRI and SacI restriction sites to replace the generic TATA box promoter of the pGL4.23 luciferase reporter construct to create the pCNR1prom-Luc (Fig 2A).pECR1C/TLuc. The ECR1 enhancer sequence was also recovered from pGEM-T easy (Nicoll et al 2012) and the minor T allele of ECR1 was re-created using site-directed mutagenesis where the template DNA was a previously established pECR1(C)Gem-Teasy construct, primers for sitedirected mutagenesis were:Production of pECR1(C)CNR1promLuc and pECR1(T)CNR1promLuc firefly luciferase reporter constructs was achieved by cloning the ECR1(C) or ECR1(T) regions from the pGEM-Teasy parent constructs using AatII and SalI sites and ligating into AatII and XhoI sites of the pCNR1prom luciferase construct, placing ECR1(C) or ECR1(T) upstream of the CNR1prom fragment to form pECR1C/TLuc (Fig 2A) pCNR1prom-LacZ. pCNR1prom was recovered from pCNR1promLuc and cloned into the NotI and SpeI sites of the LacZ reporter plasmid p1229 (10).pCNR1promLucCpG free. A CpG dinucleotide-free version of the CNR1prom-Luc reporter plasmid was created using the pCpGL-basic reporter plasmid (Klug and Rehli, 2006) and cloning the CNR1prom fragment from pCNR1promLuc reporter using BglII and NcoI sites. Plasmid methylation;Subsequently, 5μg of pCNR1promLucCpG free plasmid was methylated in a reaction containing 1x NEB buffer 2, 640μM SAM and 20U M.Sss1 enzyme (New England Biolabs) at 37°C followed by inactivation (65°C, 20mins) at various time points up to 1 hour (Full methylation). Levels of methylation were monitored using digestion by the HpaII enzyme that is
These authors contributed equally to this work. AbstractThe microbial communities resident in animal intestines are composed of multiple species that together play important roles in host development, health and disease. Due to the complexity of these communities and the difficulty of characterizing them in situ, the determinants of microbial composition remain largely unknown. Further, it is unclear for many multi-species consortia whether their species-level makeup can be predicted based on an understanding of pairwise species interactions, or whether higher-order interactions are needed to explain emergent compositions. To address this, we examine commensal intestinal microbes in larval zebrafish, initially raised germ-free to allow introduction of controlled combinations of bacterial species. Using a dissection and plating assay, we demonstrate the construction of communities of one to five bacterial species and show that the outcomes from the two-species competitions fail to predict species abundances in more complex communities. With multiple species present, inter-bacterial interactions become weaker and more cooperative, suggesting that higher-order interactions in the vertebrate gut may stabilize complex communities. 5 immune regulation [5-10] and a wide range of diseases [11][12][13][14][15][16][17][18][19][20]. 6 Despite the importance of intestinal communities, the determinants of their 7 composition remain largely unknown. A growing number of studies map the effects of 8 external perturbations, such as antibiotic drugs [21, 22] and dietary fiber [23] and 9 fat [24, 25] on the relative abundance of gut microbial species. Intrinsic inter-microbial 10interactions, however, are especially challenging to measure and are important not only 11 for shaping community composition in the absence of perturbations but also for 12 propagating species-specific perturbations to the rest of the intestinal ecosystem. 13The considerable majority of studies of the gut microbiota have been performed on 14 naturally assembled microbiomes by sequencing DNA extracted from fecal samples, an 15May 28, 2020 1/23approach that provides information about the microbial species and genes present in the 16 gut, but that imposes several limitations on the inference of inter-species interactions. 17The high diversity of natural intestinal communities, and therefore the low abundance 18 of any given species among the multitude of its fellow residents, implies that stochastic 19 fluctuations in each species' abundance will be large, easily masking true biological 20 interactions. The accuracy of inference is considerably worse if only relative, rather than 21 absolute, abundance data is available [26-29], as is typically the case in 22 sequencing-based studies. Finally, we note that fecal sampling assesses only the 23 microbes that have exited the host, which may not be representative of the intestinal 24 community [30]. 25 An alternative approach to using DNA sequencing and naturally assembled 26 host-microbiota systems is to build suc...
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