One promising approach to balance the need for fertilizer and the welfare of the environment, is the development of plants that can fix atmospheric nitrogen independently. This innovation would not only reduce the need for synthetic fertilizers and manure but also help mitigate climate change and the nitrogen crisis. To this end, we need to better study the nitroplast and how it could be introduced into other cells. <br><br>
Studies have demonstrated the insertion of bacteria into cells by engineering endosymbionts in <i>S. cerevisiae</i> using <i>E. coli</i> and <i>S. elongatus</i><ahref="#cite15">[15]</a>. Another study successfully inserted <i>Azotobacter</i> strains into <i>C. reinhardtii</i><ahref="#cite16">[16]. Building on this research, we aim to <strong>develop a reliable protocol for transplanting a nitroplast</strong> into <i>C. reinhardtii</i> and <i>S. cerevisiae</i>, as a <strong>proof-of-concept</strong> for transplantation into other eukaryotes. We will use polyethylene glycol (PEG) fusion protocols, starting out with analogous bacteria to UCYN-A (<i>Azotobacter</i> genus and <i>Cyanothece</i> ATCC51142, the closest free-living relative of UCYN-A). <br><br>
Studies have demonstrated the insertion of bacteria into cells by engineering endosymbionts in <i>S. cerevisiae</i> using <i>E. coli</i> and <i>S. elongatus</i><ahref="#cite15">[15]</a>. Another study successfully inserted <i>Azotobacter</i> strains into <i>C. reinhardtii</i><ahref="#cite16">[16]</a>. Building on this research, we aim to <strong>develop a reliable protocol for transplanting a nitroplast</strong> into <i>C. reinhardtii</i> and <i>S. cerevisiae</i>, as a <strong>proof-of-concept</strong> for transplantation into other eukaryotes. We will use polyethylene glycol (PEG) fusion protocols, starting out with analogous bacteria to UCYN-A (<i>Azotobacter</i> genus and <i>Cyanothece</i> ATCC51142, the closest free-living relative of UCYN-A). <br><br>
It has been discovered that several essential UCYN-A proteins are expressed in the host, <i>B. bigelowii</i>, and imported into the symbiont, not unlike chloroplasts and mitochondria, though to a lesser extent <ahref="#cite14">[14]</a>. Many of these proteins possess a C-terminal extension known as the “uTP” (UCYN-A Transit Peptide) <ahref="#cite14">[14]</a>. We first aim to use bioinformatic analysis to identify the characteristic <strong>motifs required for a protein to be imported into UCYN-A</strong>. For this, we will make use of host and nitroplast's genome data as well as the proteomics data published by Coale <i>et al.</i>. <br><br>
To understand the functioning of the UCYN-A import mechanism, we will attempt to <strong>identify the proteins involved in translocating</strong> host-encoded proteins into UCYN-A. First, we will locate genes in the host genome which are potentially involved in the translocation based on their similarity to proteins in other import mechanisms such as the <i>Paulinella chromatophora</i> (UCYN-A analogue for photosynthesis) protein import or chaperones that seem analogous to heat-shock proteins. These chaperones are hypothesized to bind to proteins tagged by the uTP and keep them from folding, allowing translocation through the UCYN-A membrane. Following this, we will <strong>obtain the tertiary structure of all candidate proteins</strong> using a structure prediction tool, and use <strong>docking</strong> tools to select ones that are likely to bind the previously identified transit motifs. <br><br>
The initial <i>in vivo</i> characterization of the UCYN-A transport system will involve <strong>examining the expression and localization of the UCYN-A transit peptides in the eukaryotic model organisms</strong><i>S. cerevisiae</i> and <i>C. reinhardtii</i> to test for interference by cellular processes. To this end, uTP-tagged fluorescent proteins will be expressed in <i>S. cerevisiae</i> and <i>C. reinhardtii</i>, and the constructs will be confirmed using fluorescence microscopy. <br><br>
