Team:SDU-Denmark/Tour42

OneProt

The pTet expression system and limonene synthase construct is evolved around one thing: the OneProt. We have made the pTet-OneProt construct in order for us to synthesize a nutritional protein with the correct ratio of essential amino acids and the correct ratio between essential and non-essential amino acids. The device is found as Bba_K1475000.

The protein is self-designed, so we wanted to test if the protein were expressed in E. coli K12 MG1655, by the use of Western blotting. The western blot was blottet with E. coli K12 MG1655 wild-type and E. coli expressing OneProt at different OD measures.

Figure 1: Western blot showing E. coli wild-type at OD600 at 0.3, 0.8 and 1.8 and E. coli expressing OneProt at OD600 at 0.3, 0.8, 1.8 and an overnight culture.

The protein has a 3xFLAG tag and since bonds are showing, OneProt is expressed. However, from this western blot, we cannot see if the protein has been cut, just that it is expressed.

Figure 2: Coomassie staining of E. coli expressing OneProt at early exponential phase (OD600=0.3), late exponential phase (OD600=1.5), stationary phase (OD=2.5) and an overnight culture using an empty vector as control. In order to check that we had the protein expressed in its full length, we did a coomassie stain on a SDS- page. Here we also wanted to receive information on the expression of the protein at different growth stages of E. coli. We analyzed samples from early exponential phase (OD600=0.3), late exponential phase (OD600=1.5), stationary phase (OD=2.5) and an overnight culture. As a control, E. coli with an empty vector (PSC1C3) was used.

OneProt has a molecular weight of approximately 53.7 kDa. Unfortunately, there is no clear bond at this length. However, there is a bond at approximately 25 kDa, which is not detected in the control. We cannot conclude what gives rise to the band, but it might be a cellular response to an unfolded protein.

To test what effect the expression of OneProt have on E. coli we set up a growth experiment measuring OD over time on the growth of E. coli K12 MG1655 WT, odor-free E. coli YYC912, E. coli K12 expressing OneProt and with an empty vector.

Figure 3: Growth curve illustrating the growth of E. coli K12 MG1655 WT, odor-free E. coli YYC912, E. coli K12 expressing OneProt and with an empty vector. From the growth curve, it is shown that the growth-rate of neither the strain expressing the OneProt protein nor the YYC912 strain is affected a lot compared to the E. coli K12 wild-type.

By comparing the growth curve of E. coli K12 expressing OneProt, TetR(+LVA), TetR(no LVA) and odor-free E. coli YYC912 it is seen that the growth-rate of E. coli expressing TetR is lowered compared to the other strains, which means that it might be difficult to have OneProt expressed in high amounts controlled by pTet (+/-LVA), with the TetR repressors overexpressed at present levels. However, as argued on previous page, the levels of TetR would have to be lowered, which would probably alleviate the attenuated growth-rate.

Figure 4: Growth curves showing E. coli K12 MG1655 expressing OneProt, TetR(+LVA), TetR(no LVA), wild-type and odor-free E. coli YYC912. Because OneProt is self-designed, we wanted to test if the protein has any toxicity. To do so, we fed Caenorhabditis elegans (C. elegans) with E. coli K12 MG1655 containing an empty vector and a vector expressing OneProt on separate plates. On both plates, 20 C. elegans were tested. Articles recommend using heat shock assay for 7 hours: 1 hour at 35° C followed by 1 hour at 22°C, repeated. Source: Mosbech, M., et al.: Functional Loss of Two Ceramide Synthases Elicits Autophagy-Dependent Lifespan Extension in C. elegans.: PLoS ONE, 2013. 8 vol:7. (Link)   Source: Rodriguez, M., et al.:Worms under stress: C. elegans stress response and its relevance to complex human disease and aging. Trends in Genetics, 2013. Vol: 29, 6, p. 367-374. (Link)

After approximately 5 hours no effects on C. elegans was detectable. Therefore we decided to stress C. elegans a little more, incubating them in 2 hours at 35°C followed by 1 hour at 22°C, repeated. After 7 hours, every C. elegans on both plates were alive. Thus we conclude that the protein has no toxic effect.

Figure 5: Picture of C. elegans fed with E. coli K12 MG1655 expressing OneProt.