Part:BBa_M36140:Experience

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Applications of BBa_M36140

This protein, when coupled with GvpC, induces buoyancy in E. coli. When in the ideal 1:25 ratio of GvpC to GvpA, structural gas vesicles form that decrease the density of the bacterium and cause floatation.

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Coupled with a high copy ORI number and ampicillin resistance. The promoter was attached to a glucose-repressable, rhamnose-inducible sensor. This plasmid was transformed into E.coli cells and then co-transformed with a GvpA stock to create a hybrid GvpA + GvpC E. coli strain.

OD Analysis

OD Analysis was performed to track settling patterns in a variety of control stocks and experimental stocks. A four-day trial was designed based upon the troubleshooting trial, with measurements of 1-8 M9+/- glucose, M9+/- rhamnose, LB+/- glucose, and LB+/- rhamnose taken at 0, 25, 29.75, 48, 53, and 72 hours. In this experiment, we optimized the volume of culture to 400uL—right above the reading line of the OD machine—so that an increase in OD would correlate to floating and decrease would imply sinking. After recording our data over a 72-hour time scale, we created graphs and fit linear trendlines to the data. We used the slope of these trendlines (which correlates to change in OD over hours) as a measure of the amount of settling that occurred. Generally, most of our bacteria exhibited settling (except for M9 Rhamnose cultures 4 and 8), as indicated by their negative slopes. If we compare the values of the two cultures that were supposed to exhibit floatation (M9 + Rhamnose + Hybrid and LB + Rhamnose + Hybrid), we can see that the slopes were -0.0009 and -0.0049 respectively. In comparison to the other slopes, the M9 + Rhamnose + Hybrid could potentially suggest a lesser rate of sinking. However, because the R2 values associated with our data graphs’ trendlines were extremely varied - ranging from 0.15 to 0.992 - this suggests the data procured is not consistent, thus we cannot come to a conclusion as to whether our gene was expressed or not.

Microscopy

To verify vesicle production, we decided to use light microscopy. This process consisted of us putting some of the bacteria onto slides and analyzing it via microscopy in hopes of finding vesicles. Unfortunately, due to the small size of E. Coli, and the strength of the available microscopes, we were unable to achieve sufficient magnification to properly view the inside of the cells (Figure 9, Supplementary Materials). When looking for elongation of the cells, we determined that there is no conclusive evidence about whether the size of our control Beta-10 cells was less-than or greater-than the cells containing our constructs.