MEMS micro-contact printing engines

This thesis investigates micro-contact printing (µCP) engines using micro-electro-mechanical systems (MEMS). Such engines are self-contained and do not require further optical alignment and precision manipulation equipment. Hence they provide a low-cost and accessible method of multilevel surface patterning with sub-micron resolution. Applications include the field of biotechnology where the placement of biological ligands at well controlled locations on substrates is often required for biological assays, cell studies and manipulation, or for the fabrication of biosensors. A miniaturised silicon µCP engine is designed and fabricated using a wafer-scale MEMS fabrication process and single level and bi-level µCP are successfully demonstrated. The performance of the engine is fully characterised and two actuation modes, mechanical and electrostatic, are investigated. In addition, a novel method of integrating the stamp material into the MEMS process flow by spray coating is reported. A second µCP engine formed by wafer-scale replica moulding of a polymer is developed to further drive down cost and complexity. This system carries six complementary patterns and allows six-level µCP with a layer-to-layer accuracy of 10 µm over a 5 mm x 5 mm area without the use of external aligning equipment. This is the first such report of aligned multilevel µCP. Lastly, the integration of the replica moulded engine with a hydraulic drive for controlled actuation is investigated. This approach is promising and proof of concept has been provided for single-level patterning

Ribosomal protein L20 controls expression of the Bacillus subtilis infC operon via a transcription attenuation mechanism

In contrast to Escherichia coli no molecular mechanism controlling the biosynthesis of ribosomal proteins has been elucidated in Gram-positive organisms. Here we show that the expression of the Bacillus subtilis infC-rpmI-rplT operon encoding translation factor IF3 and the ribosomal proteins L35 and L20 is autoregulated by a complex transcription attenuation mechanism. It implicates a 200-bp leader region upstream of infC which contains two conserved regulatory elements, one of which can act as a transcription terminator. Using in vitro and in vivo approaches we show that expression of the operon is regulated at the level of transcription elongation by a change in the structure of the leader mRNA which depends upon the presence of ribosomal protein L20. L20 binds to a phylogenetically conserved domain and provokes premature transcription termination at the leader terminator. Footprint and toeprint experiments support a regulatory model involving molecular mimicry between the L20-binding sites on 23S rRNA and the mRNA. Our data suggest that Nomura's model of ribosomal protein biosynthesis based on autogenous control and molecular mimicry is also valid in Gram-positive organisms

A micro-optical module for multi-wavelength addressing of trapped ions

The control of large-scale quantum information processors based on arrays of trapped ions requires a means to route and focus multiple laser beams to each of many trapping sites in parallel. Here, we combine arrays of fibres, 3D laser-written waveguides and diffractive microlenses to demonstrate the principle of a micro-optic interconnect suited to this task. The module is intended for use with an ion microtrap of 3D electrode geometry. It guides ten independent laser beams with unique trajectories to illuminate a pair of spatially separated target points. Three blue and two infrared beams converge to overlap precisely at each desired position. Typical relative crosstalk intensities in the blue are $3.6 \times 10^{-3}$ and the average insertion loss across all channels is $8~$dB. The module occupies $\sim 10^4$ times less volume than a conventional bulk-optic equivalent and is suited to different ion species

Radio-frequency microplasmas with energies suited to in situ selective cleaning of surface adsorbates in ion microtraps

We have demonstrated a capacitively-coupled, radio-frequency (RF) microplasma inside the 3D electrode structure of an ion microtrap device. For this work, devices with an inter-electrode distance of 340 μm were used. The microplasmas were operated at Ω RF /2π = 23 MHz, in both He and He:N2 gas mixtures, over a range of RF amplitudes (140–220 V) and pressures (250–910 mbar). Spectroscopic analysis of the He I 667 nm and Hα 656 nm emission lines yielded the gas temperature and electron density, which enabled calculation of the mean ion bombardment energy. For the range of operating parameters studied, we calculated mean He+ energies to be between 0.3 and 4.1 eV. While these energies are less than the threshold for He sputtering of hydrocarbon adsorbates on Au, we calculate that the high energy tail of the distribution should remove adsorbate monolayers in as little as 1 min of processing. We also calculate that the distribution is insufficiently energetic to have any significant effect on the Au electrode surface within that duration. Our results suggest that the microplasma technique is suited to in situ selective removal of surface adsorbates from ion microtrap electrodes

Magneto-inductive magnetic resonance imaging duodenoscope

A magnetic resonance imaging (MRI) duodenoscope is demonstrated, by combining non- magnetic endoscope components with a thin-film receiver based on a magneto-inductive waveguide. The waveguide elements consist of figure-of-eight shaped inductors formed on either side of a flexible substrate and parallel plate capacitors that use the substrate as a dielectric. Operation is simulated using equivalent circuit models and by computation of two- and three-dimensional sensitivity patterns. Circuits are fabricated for operation at 127.7 MHz by double-sided patterning of copper-clad Kapton and assembled onto non-magnetic flexible endoscope insertion tubes. Operation is verified by bench testing and by 1 H MRI at 3T using phantoms. The receiver can form a segmented coaxial image along the length of the endoscope, even when bent, and shows a signal-to-noise-ratio advantage over a surface array coil up to three times the tube diameter at the tip. Initial immersion imaging experiments have been carried out and confirm an encouraging lack of sensitivity to RF heating

Rapid changes in gene expression: DNA determinants of promoter regulation by the concentration of the transcription initiating NTP in Bacillus subtilis

In bacteria, rapid changes in gene expression can be achieved by affecting the activity of RNA polymerase with small molecule effectors during transcription initiation. An important small molecule effector is the initiating nucleoside triphosphate (iNTP). At some promoters, an increasing iNTP concentration stimulates promoter activity, while a decreasing concentration has the opposite effect. Ribosomal RNA (rRNA) promoters from Gram-positive Bacillus subtilis are regulated by the concentration of their iNTP. Yet, the sequences of these promoters do not emulate the sequence characteristics of [iNTP]-regulated rRNA promoters of Gram-negative Escherichia coli. Here, we identified the 3′-promoter region, corresponding to the transcription bubble, as key for B. subtilis rRNA promoter regulation via the concentration of the iNTP. Within this region, the conserved −5T (3 bp downstream from the −10 hexamer) is required for this regulation. Moreover, we identified a second class of [iNTP]-regulated promoters in B. subtilis where the sequence determinants are not limited to the transcription bubble region. Overall, it seems that various sequence combinations can result in promoter regulation by [iNTP] in B. subtilis. Finally, this study demonstrates how the same type of regulation can be achieved with strikingly different promoter sequences in phylogenetically distant species

A search for small noncoding RNAs in Staphylococcus aureus reveals a conserved sequence motif for regulation

Bioinformatic analysis of the intergenic regions of Staphylococcus aureus predicted multiple regulatory regions. From this analysis, we characterized 11 novel noncoding RNAs (RsaA‐K) that are expressed in several S. aureus strains under different experimental conditions. Many of them accumulate in the late-exponential phase of growth. All ncRNAs are stable and their expression is Hfq-independent. The transcription of several of them is regulated by the alternative sigma B factor (RsaA, D and F) while the expression of RsaE is agrA-dependent. Six of these ncRNAs are specific to S. aureus, four are conserved in other Staphylococci, and RsaE is also present in Bacillaceae. Transcriptomic and proteomic analysis indicated that RsaE regulates the synthesis of proteins involved in various metabolic pathways. Phylogenetic analysis combined with RNA structure probing, searches for RsaE‐mRNA base pairing, and toeprinting assays indicate that a conserved and unpaired UCCC sequence motif of RsaE binds to target mRNAs and prevents the formation of the ribosomal initiation complex. This study unexpectedly shows that most of the novel ncRNAs carry the conserved C−rich motif, suggesting that they are members of a class of ncRNAs that target mRNAs by a shared mechanism

Sequence-Based Analysis Uncovers an Abundance of Non-Coding RNA in the Total Transcriptome of Mycobacterium tuberculosis

RNA sequencing provides a new perspective on the genome of Mycobacterium tuberculosis by revealing an extensive presence of non-coding RNA, including long 5’ and 3’ untranslated regions, antisense transcripts, and intergenic small RNA (sRNA) molecules. More than a quarter of all sequence reads mapping outside of ribosomal RNA genes represent non-coding RNA, and the density of reads mapping to intergenic regions was more than two-fold higher than that mapping to annotated coding sequences. Selected sRNAs were found at increased abundance in stationary phase cultures and accumulated to remarkably high levels in the lungs of chronically infected mice, indicating a potential contribution to pathogenesis. The ability of tubercle bacilli to adapt to changing environments within the host is critical to their ability to cause disease and to persist during drug treatment; it is likely that novel post-transcriptional regulatory networks will play an important role in these adaptive responses