Team:Stanford-Brown/VenusLife/Biosensing

Remote Biosensing

In order to explore hypotheses of aerosolized Venusian life, we must implement a means to observe growth in situ. Since we are ultimately interested in whether or not organisms can inhabit a certain (extraterrestrial) niche, our organisms must exhibit a reporter for growth, not survivability. Since bacteria can sporulate or enter bacteriostasis under stress, thereby surviving without growing or reproducing, it is important to distinguish bacterial growth or reproduction as an indicator of successful inhabitation.

It is necessary that researchers can measure such a reporter without interfering with an ongoing experiment. In the case of our project, which utilizes a suspension chamber, an adapted Millikan apparatus, to aerosolize bacteria, it would be impossible to observe the aerosolized bacteria except through a viewing scope. Normal techniques of quantifying growth and replication, such as FACS or measuring optical density via spectrophotometry, would not be useful. As such, we decided to design and create a fluorescent cell-cycle dependent reporter to enable optical analysis of organisms in situ. Such a reporter transcends use in this isolated experiment in the lab; researchers using organisms that express this reporter can use fluorescence to measure growth rates without disturbing their sample.

In order to engineer this cell-cycle dependent reporter, we first require a cell-cycle dependent promoter. We identified two from literature that we describe at this time:

polA

DnaA is the central initiator of DNA replication in E. coli and other prokaryotic organisms, but importantly, it also functions as a transcription factor that can suppress or activate transcription of genes by binding to the DnaA box, a 9bp consensus sequence (Messer 1997). Since DnaA expression is dependent on the growth conditions of the cell (Chiaramello 1990), genes regulated by DnaA are transitively growth dependent. One such DnaA-dependent gene is polA, which codes for DNA Polymerase I, active in DNA replication (Quiñones 1997).

nrd

The nrd operon in E. coli expresses ribonucleotide reductase, an enzyme that reduces ribonucleotides into deoxyribonucleotides and is involved in bacterial cell cycle. The promoter for this operon was found to begin activation during initiation of DNA replication and is cell-cycle dependent (Sun 1992). In order to explore hypotheses of aerosolized Venusian life, we must implement a means to observe growth in situ. Since we are ultimately interested in whether or not organisms can inhabit a certain (extraterrestrial) niche, our organisms must exhibit a reporter for growth, not survivability. Since bacteria can sporulate or enter bacteriostasis under stress, thereby surviving without growing or reproducing, it is important to distinguish bacterial growth or reproduction as an indicator of successful inhabitation.

The nrd promoter also contains several DnaA boxes (Messer 1997); however, this does not seem to necessitate that nrd is related to DNA synthesis rate, as DnaA indirectly controls cell division (Messer 2002).

These promoters, once isolated from their host E. coli genome and reconstituted synthetically, drive expression of fluorescent proteins such as GFP (green fluorescent protein) in our reporter. The transformation of this device into E. coli confers this biological tool that allows for in situ measurement of growth.

Results

In order to create a cell-cycle dependent reporter, we first need a cell-cycle dependent promoter. Since DnaA not only plays a significant role in initiation of DNA replication but also serves as a transcription factor for fission-related genes, we identified a library of genes transcriptionally activated by DnaA in Escherichia coli and Bacillus subtilis. Using colony PCR, we isolated the promoters of these genes (parts K847210, K847211, K847212, K847213) and ligated them to E0840 from the Endy lab.