Team:NAWI Graz/Results

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The following results show that both of our constructs work nearly as expected. The alx-mNeonGreen construct shows, that it can be induced by change from pH 7.0 to pH above 8 and fluorescence was measured 20 minutes after this pH shift. The asr-mCardinal construct was inducible by change from pH 7.0 to pH 5.0 and lower but no fluorescence was detected. The promoter itself was working as expected and we were able to detect the 6xHis-tagged mCardinal protein in cultures at pH 5.0 through a Western Blot.

pH-Sensing

Alkaline induced expression

To test if our pH sensitive constructs express the fluorescence proteins, we cultivated the bacteria hosting the plasmids in LPM with pH 7 overnight. For expression control of our alx-mNeonGreen construct, we inoculated 20 ml LPM with pH 7.0, 8.0 and 8.5 to an OD₆₀₀ of 0.2 using the overnight culture. After 20 and 40 minutes 1 ml of each culture was taken and adjusted to the lowest OD₆₀₀ of the three samples to standardize the OD₆₀₀. This was necessary as growth at pH 8.0 and 8.5 was significantly slower than at pH 7.0. The diluted samples were used to measure fluorescence (excitation 490 nm, emission 520 nm) and again OD₆₀₀ with our plate reader. Three aliquots of each sample were measured (n=3), fluorescence was divided by OD₆₀₀ and averages of the three aliquots were taken to obtain the data shown in Figure 1.

Fig 1. Alx controlled mNeonGreen Expression: Culture medium was buffered to pH 7, 8 and 8.5 and inoculated to an OD₆₀₀ of 0.2. After 20 and 40 minutes, aliquots were taken and fluorescence (excitation 490 nm, absorbance 520 nm) and OD₆₀₀ were measured with the plate reader (n=3).

As shown in Fig. 1 fluorescence increases at pH 8.0 and 8.5 at both times compared to a standard culture at pH 7.0. At pH 7.0 cultures still show high fluorescence values with no difference between cultures with our plasmid or cultures without any DNA coding for mNeonGreen (Data not shown). This leads to the conclusion that bacteria on their own emit at ~520 nm when stimulated at 490 nm. Still fluorescence is significantly increased in all cultures at higher pH with a maximum of fluorescence at pH 8.5 after 20 min. This increase in fluorescence allows to distinguish between induced and uninduced. Hence we could show that our construct works as planned. The next step would be to test how much base (NaOH) would be needed to change the pH of a defined volume of culture with pH 7.0 to pH 8.5. This data would enable us to use the construct to control the robot. Unfortunately the summer was to short to complete this task.

Acid induced expression

To test acid induced expression of mCardinal controlled by the asr promoter (our asr-mCardinal construct), we tried the same protocol we used for the alx-mNeonGreen but with pH 7.0, 5.0 and 4.5. Unfortunately this did not work out, because, as we discovered later, expression works, but mCardinal, a fluorescence protein with extreme fast maturation time, is designed for expression in mammalian¹ cells and does not show any fluorescence when expressed in E. coli. Therefore we investigated the expression on the protein level with Western blotting. To do so we cultivated bacteria containing the construct overnight in LPM with pH 7.0 and used this culture to inoculate 100 ml LPM with pH 7.0, 5.0 and 4.5 to an OD₆₀₀ of 0.1. The cultures were incubated at 37°C. Before inoculating, after 1.5, 2.0 and 2.5 hours 3 OD units were taken of each culture, pelleted and cooled on ice. After all samples were collected they were prepared for SDS gel electrophoresis by sonicating, mixing with loading buffer and heating to 95°C. Samples and pageruler protein standard were separated on a SDS gel and blotted on a nitrocellulose membrane. After washing and incubating with anti-6xHis antibodies tagged with horse radish peroxidase, detection was performed with Thermo Scientific^TM SuperSignal^TM West Pico Chemiluminescent Substrate.

Fig 2. asr controlled expression of mCardinal: Overlay of the membrane showing the PageRuler prestained protein ladder (S) and a picture showing the signal of the horse radish peroxidase substrate. Lanes from left to right, protein ladder, inoculation culture with pH 7.0, the three samples, pH 7.0, 5.0, 4.5, after 1.5 h (t1), after 2 h (t2) and after 2.5 h (t3).

All detected signals seen in Fig. 2 are the same height as the 30 kDa band of the standard, correlating with the size of mCardinal plus TEV-site, f-degron and the 6xHis-tag, with an approximate weight of 29.9 kDa. There is no signal in any sample with pH 7.0 leading to the conclusion that asr is not active at pH 7.0. At pH 5.0 and 4.5 there are bands of different intensity at all time points, hence asr is active at these pH and mCardinal is expressed. This is proof that our construct does work. Also expression is higher at pH 5.0 than at 4.5 especially after 2h (t2) and 2.5 h (t3). To verify these results and use the construct for our robot, a different fluorescence protein can be inserted in the construct and the new construct could be tested and used like the alx construct above.

1. Chu, J. et al. Non-invasive intravital imaging of cellular differentiation with a bright red-excitable fluorescent protein. Nature methods 11, 572–578 (2014).