A Two-Layer Gene Circuit for Decoupling Cell Growth from Metabolite Production

SummaryWe present a synthetic gene circuit for decoupling cell growth from metabolite production through autonomous regulation of enzymatic pathways by integrated modules that sense nutrient and substrate. The two-layer circuit allows Escherichia coli to selectively utilize target substrates in a mixed pool; channel metabolic resources to growth by delaying enzymatic conversion until nutrient depletion; and activate, terminate, and re-activate conversion upon substrate availability. We developed two versions of controller, both of which have glucose nutrient sensors but differ in their substrate-sensing modules. One controller is specific for hydroxycinnamic acid and the other for oleic acid. Our hydroxycinnamic acid controller lowered metabolic stress 2-fold and increased the growth rate 2-fold and productivity 5-fold, whereas our oleic acid controller lowered metabolic stress 2-fold and increased the growth rate 1.3-fold and productivity 2.4-fold. These results demonstrate the potential for engineering strategies that decouple growth and production to make bio-based production more economical and sustainable

Engineering antisense oligonucleotides as a tool in synthetic biology.

Synthetic biology is an interdisciplinary field that comprises of the use of engineering principles to design biological systems. A distinct feature of synthetic biology is the emphasis of control over an engineered system. In this study, we used antisense oligonucleotides that did not contain any backbone modifications to repress the effects of basal transcription of an inducible promoter, acrAp. Two types of constructs, RFP and GFP were each attached to six sets of antisense oligonucleotides with different lengths. All constructs were then induced with 6 different inducer concentrations. The maximum repression level of 42.2% was achieved by a GFP construct with one-fifth GFP antisense oligonucleotides in the presence of 0.2 % (w/v) arabinose. However, results obtained were not translatable between RFP and GFP constructs. Cells with half, one-third and one-fifth RFP antisense oligonucleotides showed repression activity. In contrast, all the six GFP antisense oligonucleotides were able to lower translation level in their respective cells. This discrepancy between the two sets of results might be due to the design of the constructs. The RFP plasmid had a native RBS while RBS in GFP plasmid was inserted. Hence, this had caused a six base pair scar between the RBS and GFP coding gene.Bachelor of Science in Biological Science

The binding interface of kindlin-2 and ILK involves Asp344/Asp352/Thr356 in kindlin-2 and Arg243/Arg334 in ILK

Focal adhesion (FA) proteins, kindlin‐2 and integrin‐linked kinase (ILK), regulate cell adhesion and migration. ILK interacts with and promotes kindlin‐2 targeting to FAs. Leu353 and Leu357 in kindlin‐2 have been reported to be important for the interaction between kindlin‐2 and ILK. However, the binding interface between kindlin‐2 and ILK remains unclear. Using molecular modeling and molecular dynamics simulations, we show that Asp344, Asp352, and Thr356 in kindlin‐2 and Arg243 and Arg334 in ILK kinase domain (KD) are important in kindlin‐2/ILK complex formation. Mutations that disrupt these interactions abrogate kindlin‐2 and ILK colocalization in HeLa cells. The interactions are direct based on data from pull‐down assays using purified recombinant kindlin‐2 F2‐pleckstrin homology and ILK KDs. These data provide additional insights into the binding interface between kindlin‐2 and ILK.ASTAR (Agency for Sci., Tech. and Research, S’pore)MOE (Min. of Education, S’pore)Accepted versio

Biosynthesis of Commodity Chemicals From Oil Palm Empty Fruit Bunch Lignin

10.3389/fmicb.2021.663642FRONTIERS IN MICROBIOLOGY1

An AR-ERG transcriptional signature defined by long-range chromatin interactomes in prostate cancer cells.

The aberrant activities of transcription factors such as the androgen receptor (AR) underpin prostate cancer development. While the AR cis-regulation has been extensively studied in prostate cancer, information pertaining to the spatial architecture of the AR transcriptional circuitry remains limited. In this paper, we propose a novel framework to profile long-range chromatin interactions associated with AR and its collaborative transcription factor, erythroblast transformation-specific related gene (ERG), using chromatin interaction analysis by paired-end tag (ChIA-PET). We identified ERG-associated long-range chromatin interactions as a cooperative component in the AR-associated chromatin interactome, acting in concert to achieve coordinated regulation of a subset of AR target genes. Through multifaceted functional data analysis, we found that AR-ERG interaction hub regions are characterized by distinct functional signatures, including bidirectional transcription and cotranscription factor binding. In addition, cancer-associated long noncoding RNAs were found to be connected near protein-coding genes through AR-ERG looping. Finally, we found strong enrichment of prostate cancer genome-wide association study (GWAS) single nucleotide polymorphisms (SNPs) at AR-ERG co-binding sites participating in chromatin interactions and gene regulation, suggesting GWAS target genes identified from chromatin looping data provide more biologically relevant findings than using the nearest gene approach. Taken together, our results revealed an AR-ERG-centric higher-order chromatin structure that drives coordinated gene expression in prostate cancer progression and the identification of potential target genes for therapeutic intervention