Matching Items (2)
Filtering by
- All Subjects: Quorum Sensing
- All Subjects: Transfection
- Creators: Haynes, Karmella
- Creators: Muller, Ryan
- Member of: Barrett, The Honors College Thesis/Creative Project Collection
- Status: Published
Description
Currently in synthetic biology only the Las, Lux, and Rhl quorum sensing pathways have been adapted for broad engineering use. Quorum sensing allows a means of cell to cell communication in which a designated sender cell produces quorum sensing molecules that modify gene expression of a designated receiver cell. While useful, these three quorum sensing pathways exhibit a nontrivial level of crosstalk, hindering robust engineering and leading to unexpected effects in a given design. To address the lack of orthogonality among these three quorum sensing pathways, previous scientists have attempted to perform directed evolution on components of the quorum sensing pathway. While a powerful tool, directed evolution is limited by the subspace that is defined by the protein. For this reason, we take an evolutionary biology approach to identify new orthogonal quorum sensing networks and test these networks for cross-talk with currently-used networks. By charting characteristics of acyl homoserine lactone (AHL) molecules used across quorum sensing pathways in nature, we have identified favorable candidate pathways likely to display orthogonality. These include Aub, Bja, Bra, Cer, Esa, Las, Lux, Rhl, Rpa, and Sin, which we have begun constructing and testing. Our synthetic circuits express GFP in response to a quorum sensing molecule, allowing quantitative measurement of orthogonality between pairs. By determining orthogonal quorum sensing pairs, we hope to identify and adapt novel quorum sensing pathways for robust use in higher-order genetic circuits.
ContributorsMuller, Ryan (Author) / Haynes, Karmella (Thesis director) / Wang, Xiao (Committee member) / Barrett, The Honors College (Contributor) / School of Mathematical and Statistical Sciences (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
The use of microelectrode arrays (MEA) to electroporate cells is now a reliable way of transfecting RNA interfering substances with high viability and efficiency. However, as the 50-200 micron electrodes are coated with many cells, there are differences in both viability and efficiency between the outside and inside of the electrode. This is due to the field created by the electrode, which has higher intensities toward the outside and lower intensities toward the middle. In order to get the electric field to spread in a more even manner, an "Anodisc" inorganic membrane seeded with cells was placed on the MEA to act as a buffer to the electric fields. One hundred percent transfection efficiency on live cells was found on one sample, though there were problems encountered along the experimental process that introduced error into the results, some of which included the inability for cells to grow to high levels of confluency on the Anodisc as well as the inverted imaging technique used on the opaque disc.
ContributorsDonnelly, Kyle Robert (Author) / Muthuswamy, Jitendran (Thesis director) / Haynes, Karmella (Committee member) / LaBelle, Jeffrey (Committee member) / Barrett, The Honors College (Contributor) / Harrington Bioengineering Program (Contributor)
Created2013-05