L-homoserine is a pivotal intermediate in the carbon and nitrogen metabolism of
E. coli.
However, this non-canonical amino acid cannot be used as a nitrogen source for growth. Furthermore, growth of ...this bacterium in a synthetic media is potently inhibited by L-homoserine. To understand this dual effect, an adapted laboratory evolution (ALE) was applied, which allowed the isolation of a strain able to grow with L-homoserine as the nitrogen source and was, at the same time, desensitized to growth inhibition by this amino acid. Sequencing of this evolved strain identified only four genomic modifications, including a 49 bp truncation starting from the stop codon of
thrL
. This mutation resulted in a modified
thrL
locus carrying a
thrL*
allele encoding a polypeptide 9 amino acids longer than the
thrL
encoded leader peptide. Remarkably, the replacement of
thrL
with
thrL*
in the original strain MG1655 alleviated L-homoserine inhibition to the same extent as strain 4E, but did not allow growth with this amino acid as a nitrogen source. The loss of L-homoserine toxic effect could be explained by the rapid conversion of L-homoserine into threonine
via
the
thrL*-
dependent transcriptional activation of the threonine operon
thrABC
. On the other hand, the growth of
E. coli
on a mineral medium with L-homoserine required an activation of the threonine degradation pathway II and glycine cleavage system, resulting in the release of ammonium ions that were likely recaptured by NAD(P)-dependent glutamate dehydrogenase. To infer about the direct molecular targets of L-homoserine toxicity, a transcriptomic analysis of wild-type MG1655 in the presence of 10 mM L-homoserine was performed, which notably identified a potent repression of locomotion-motility-chemotaxis process and of branched-chain amino acids synthesis. Since the magnitude of these effects was lower in a
ΔthrL
mutant, concomitant with a twofold lower sensitivity of this mutant to L-homoserine, it could be argued that growth inhibition by L-homoserine is due to the repression of these biological processes. In addition, L-homoserine induced a strong upregulation of genes in the sulfate reductive assimilation pathway, including those encoding its transport. How this non-canonical amino acid triggers these transcriptomic changes is discussed.
L-homoserine is a pivotal intermediate in the carbon and nitrogen metabolism of
However, this non-canonical amino acid cannot be used as a nitrogen source for growth. Furthermore, growth of this ...bacterium in a synthetic media is potently inhibited by L-homoserine. To understand this dual effect, an adapted laboratory evolution (ALE) was applied, which allowed the isolation of a strain able to grow with L-homoserine as the nitrogen source and was, at the same time, desensitized to growth inhibition by this amino acid. Sequencing of this evolved strain identified only four genomic modifications, including a 49 bp truncation starting from the stop codon of
. This mutation resulted in a modified
locus carrying a
allele encoding a polypeptide 9 amino acids longer than the
encoded leader peptide. Remarkably, the replacement of
with
in the original strain MG1655 alleviated L-homoserine inhibition to the same extent as strain 4E, but did not allow growth with this amino acid as a nitrogen source. The loss of L-homoserine toxic effect could be explained by the rapid conversion of L-homoserine into threonine
the
dependent transcriptional activation of the threonine operon
. On the other hand, the growth of
on a mineral medium with L-homoserine required an activation of the threonine degradation pathway II and glycine cleavage system, resulting in the release of ammonium ions that were likely recaptured by NAD(P)-dependent glutamate dehydrogenase. To infer about the direct molecular targets of L-homoserine toxicity, a transcriptomic analysis of wild-type MG1655 in the presence of 10 mM L-homoserine was performed, which notably identified a potent repression of locomotion-motility-chemotaxis process and of branched-chain amino acids synthesis. Since the magnitude of these effects was lower in a
mutant, concomitant with a twofold lower sensitivity of this mutant to L-homoserine, it could be argued that growth inhibition by L-homoserine is due to the repression of these biological processes. In addition, L-homoserine induced a strong upregulation of genes in the sulfate reductive assimilation pathway, including those encoding its transport. How this non-canonical amino acid triggers these transcriptomic changes is discussed.
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•We obtain homogeneous DNA transfer in μ-contact printing by dynamic inking process.•The speed of the triple line is found critical in the quality of dynamic inking process.•DNA ...inking time in μ-contact printing (μCP) is not key to inking quality.•DNA consumption is reduced 10-folds.•This method paves the way to μCP as an industrial solution for biochips fabrication.
Microcontact printing (μCP) is used as a patterning technique to produce simple, rapid and cost-effective DNA microarrays. The accuracy of the final transferred pattern drastically depends on the inking step. The usual way to ink a PDMS stamp by droplet deposition of labeled biomolecules using a pipette, results in irregular transfer of the biomolecules on the chip surface and leads to poor and irreproducible fluorescent signals. These drawbacks are likely due to irregular ‘coating’ of the biomolecules on the PDMS stamp. In this work, a novel approach for inking PDMS with DNA molecules is presented. It is based on the continuous displacement of the meniscus formed by the inking solution over the surface of the stamp. When compared with the conventional technique, this dynamic PDMS inking method proved to be very reproducible for producing regular prints/spots on a functionalized glass slide, and this method could be easily extrapolated at an industrial scale.
Microarrays are established research tools for genotyping, expression profiling, or molecular diagnostics in which DNA molecules are precisely addressed to the surface of a solid support. This study ...assesses the fabrication of low-density oligonucleotide arrays using an automated microcontact printing device, the InnoStamp 40(®). This automate allows a multiplexed deposition of oligoprobes on a functionalized surface by the use of a MacroStamp(TM) bearing 64 individual pillars each mounted with 50 circular micropatterns (spots) of 160 µm diameter at 320 µm pitch. Reliability and reuse of the MacroStamp(TM) were shown to be fast and robust by a simple washing step in 96% ethanol. The low-density microarrays printed on either epoxysilane or dendrimer-functionalized slides (DendriSlides) showed excellent hybridization response with complementary sequences at unusual low probe and target concentrations, since the actual probe density immobilized by this technology was at least 10-fold lower than with the conventional mechanical spotting. In addition, we found a comparable hybridization response in terms of fluorescence intensity between spotted and printed oligoarrays with a 1 nM complementary target by using a 50-fold lower probe concentration to produce the oligoarrays by the microcontact printing method. Taken together, our results lend support to the potential development of this multiplexed microcontact printing technology employing soft lithography as an alternative, cost-competitive tool for fabrication of low-density DNA microarrays.
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