3 research outputs found
Printing of Patterned, Engineered <i>E. coli</i> Biofilms with a Low-Cost 3D Printer
Biofilms
can grow on virtually any surface available, with impacts ranging
from endangering the lives of patients to degrading unwanted water
contaminants. Biofilm research is challenging due to the high degree
of biofilm heterogeneity. A method for the production of standardized,
reproducible, and patterned biofilm-inspired materials could be a
boon for biofilm research and allow for completely new engineering
applications. Here, we present such a method, combining 3D printing
with genetic engineering. We prototyped a low-cost 3D printer that
prints bioink, a suspension of bacteria in a solution of alginate
that solidifies on a calcium-containing substrate. We 3D-printed <i>Escherichia coli</i> in different shapes and in discrete layers,
after which the cells survived in the printing matrix for at least
1 week. When printed bacteria were induced to form curli fibers, the
major proteinaceous extracellular component of <i>E. coli</i> biofilms, they remained adherent to the printing substrate and stably
spatially patterned even after treatment with a matrix-dissolving
agent, indicating that a biofilm-mimicking structure had formed. This
work is the first demonstration of patterned, biofilm-inspired living
materials that are produced by genetic control over curli formation
in combination with spatial control by 3D printing. These materials
could be used as living, functional materials in applications such
as water filtration, metal ion sequestration, or civil engineering,
and potentially as standardizable models for certain curli-containing
biofilms
SMARCAD1-mediated active replication fork stability maintains genome integrity
The stalled fork protection pathway mediated by breast cancer 1/2 (BRCA1/2) proteins is critical for replication fork stability. However, it is unclear whether additional mechanisms are required to maintain replication fork stability. We describe a hitherto unknown mechanism, by which the SWI/SNF-related matrix-associated actindependent regulator of chromatin subfamily-A containing DEAD/H box-1 (SMARCAD1) stabilizes active replication forks, that is essential to maintaining resistance towards replication poisons. We find that SMARCAD1 prevents accumulation of 53BP1-associated nucleosomes to preclude toxic enrichment of 53BP1 at the forks. In the absence of SMARCAD1, 53BP1 mediates untimely dissociation of PCNA via the PCNA-unloader ATAD5, causing frequent fork stalling, inefficient fork restart, and accumulation of single-stranded DNA. Although loss of 53BP1 in SMARCAD1 mutants rescues these defects and restores genome stability, this rescued stabilization also requires BRCA1-mediated fork protection. Notably, fork protection-challenged BRCA1-deficient naïve- or chemoresistant tumors require SMARCAD1-mediated active fork stabilization to maintain unperturbed fork progression and cellular proliferation
Reproducibility of fluorescent expression from engineered biological constructs in E. coli
We present results of the first large-scale interlaboratory study carried out in synthetic biology, as part of the 2014 and 2015 International Genetically Engineered Machine (iGEM) competitions. Participants at 88 institutions around the world measured fluorescence from three engineered constitutive constructs in E. coli. Few participants were able to measure absolute fluorescence, so data was analyzed in terms of ratios. Precision was strongly related to fluorescent strength, ranging from 1.54-fold standard deviation for the ratio between strong promoters to 5.75-fold for the ratio between the strongest and weakest promoter, and while host strain did not affect expression ratios, choice of instrument did. This result shows that high quantitative precision and reproducibility of results is possible, while at the same time indicating areas needing improved laboratory practices.Peer reviewe