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. Please note that this part was characterized with an illegal XbaI site between the promoter and RBS, this has no influence on the function of the brick, the XbaI site was later removed and the brick has been submitted to the registry.

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 the 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 strain expressing OneProt at presents levels doesn't display toxic effects on C. elegans. This suggest that there are no obvious toxic effects towards eukaryotic cells such as human cells. However, the resulst are far from conclusive, and extensive testing is necessarry to confirm this suggestion.

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