3 research outputs found
A Solid-Phase Platform for Combinatorial and Scarless Multipart Gene Assembly
With
the emergence of standardized genetic modules as part of the synthetic
biology toolbox, the need for universal and automatable assembly protocols
increases. Although several methods and standards have been developed,
these all suffer from drawbacks such as the introduction of scar sequences
during ligation or the need for specific flanking sequences or <i>a priori</i> knowledge of the final sequence to be obtained.
We have developed a method for scarless ligation of multipart gene
segments in a truly sequence-independent fashion. The big advantage
of this approach is that it is combinatorial, allowing the generation
of all combinations of several variants of two or more modules to
be ligated in less than a day. This method is based on the ligation
of single-stranded or double-stranded oligodeoxynucleotides (ODN)
and PCR products immobilized on a solid support. Different settings
were tested to optimize the solid-support ligation. Finally, to show
proof of concept for this novel multipart gene assembly platform a
small library of all possible combinations of 4 BioBrick modules was
generated and tested
Application of Black Silicon for Nanostructure-Initiator Mass Spectrometry
Nanostructure-initiator
mass spectrometry (NIMS) is a matrix-free
desorption/ionization technique with high sensitivity for small molecules.
Surface preparation has relied on hydrofluoric acid (HF) electrochemical
etching which is undesirable given the significant safety controls
required in this specialized process. In this study, we examine a
conventional and widely used process for producing black silicon based
on sulfur hexafluoride/oxygen (SF<sub>6</sub>/O<sub>2</sub>) inductively
coupled plasma (ICP) etching at cryogenic temperatures and we find
it to be suitable for NIMS. A systematic study varying parameters
in the plasma etching process was performed to understand the relationship
of black silicon morphology and its sensitivity as a NIMS substrate.
The results suggest that a combination of higher silicon temperature
and oxygen flow rate gives rise to the formation of black silicon
with fine pillar structures, whose aspect ratio are ∼8.7 and
depth are <1 μm resulting in higher NIMS sensitivity which
is attributed to surface restructuring caused by their low melting
point upon laser irradiation. Interestingly, we find selectivity of
these black silicon substrates to different analytes depending on
the etching parameters. Though, the sensitivity of the dry etching
process is lower than the traditional “wet” electrochemical
etching process, it is suitable for many applications and is prepared
using conventional equipment without the use of HF
DNA-Linked Enzyme-Coupled Assay for Probing Glucosyltransferase Specificity
Traditional enzyme characterization
methods are low-throughput
and therefore limit engineering efforts in synthetic biology and biotechnology.
Here, we propose a DNA-linked enzyme-coupled assay (DLEnCA) to monitor
enzyme reactions in a high-throughput manner. Throughput is improved
by removing the need for protein purification and by limiting the
need for liquid chromatography mass spectrometry (LCMS) product detection
by linking enzymatic function to DNA modification. We demonstrate
the DLEnCA methodology using glucosyltransferases as an illustration.
The assay utilizes cell free transcription/translation systems to
produce enzymes of interest, while UDP-glucose and T4-β-glucosyltransferase
are used to modify DNA, which is detected postreaction using qPCR
or a similar means of DNA analysis. OleD and two glucosyltransferases
from <i>Arabidopsis</i> were used to verify the assay’s
generality toward glucosyltransferases. We further show DLEnCA’s
utility by mapping out the substrate specificity for these enzymes