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/Discussion

Our constructs displayed fluorescence when transformed into NEB-5alpha competent cells. While nrd-E0840 displayed sufficient fluorescence as verified by fluorescent microscopy, the original polA-E0840 construct (which uses mut3b GFP) exhibited low expression. The polA promoter was therefore digested with EcoRI and SpeI then ligated into plasmid pNCS containing a RBS, Clover, and a terminator. Clover is a highly engineered green fluorescent protein that exhibits extreme brightness (Lam 2012). This sufficiently bright polA-Clover construct, along with the original nrd-E0840 construct, are displayed in Figure 1.

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Figure 1 indicates that the sequences we ligated into BBa_E0840 constitute promoter sequences. Since E0840 consists of a ribosomal binding site (RBS), gene for GFP, and terminator, without a promoter it will not produce fluorescence. Since ligation resulted in fluorescence and negative control (E0840 alone) displays no leaky fluorescence, we can conclude that we successfully isolated promoters for nrd and polA.

nrd is cell-division dependent

The fluorescence reporter strain is mut3b GFP, which has a half-life of 33 hours. Since cell-cycle is on an order far less than 33 hours (~20 minutes), the fluorescence should not significantly decrease during the 1-2 hours of the microscope or bulk assays. Therefore, we expect a stepwise function in fluorescence readings (RFU). This matches the fluorescence reflected in the nrd bulk assay.

When the fluorescence reading is divided against cell density, we expect a roughly sinusoidal graph: fluorescence increases as initiation of cell replication begins, then decreases as optical density increases at a greater rate than expression of fluorescence. The plotted ratio data follows such sinusoidal behavior; replication events are clearly visible right before t=20 and t=40, suggesting that promoter activity is cell-cycle dependent.

polA is DNA-replication dependent

Under optimal conditions, K12 E. coli will replicate its DNA every 10 minutes (ONE OF THOSE SOURCES). Our results show oscillations at around a 10 minute period. Given that we grew our strains in fresh medium (LB) in a shaking 37C incubator, which constitutes near-optimal conditions, we can conclude that we expect ~10 minute period oscillations. From this we determine that the results suggest that our polA promoter exhibits DNA-replication dependence.