Another interesting phenome was that some mEos loaded by autotransporter showed the departing tendency from the outer membrane of bacteria(fig.5). Allowing for the secretion function of autotransporter, we thought that the departing-like fluorescent protein may be excreted by the autotransporter, suggesting that the system may be not very stable. However, back to our project, as long as the VHb gathered around the bacteria, it could collect oxygen to increase the local oxygen concentration then to benefit the growth of bacteria. Thus, the autotransporter could be applied to our project.

Test for OxygenMAX system

  We constructed intracellular expressing VHb vectors under the control of two promoters with different strength(J23104 and J23110), and the surface displaying VHb via INP vector under the control of J23104 promoter(fig.6). We transforms the three constructs to E.coli(DH5α) and measured their growth curve.

  Firstly we compared the growth curve of intracellular expressing VHb bacteria and empty vector transformed bacteria to see if the VHb works(fig.7). From the result we can see that both the two VHb expressed bacteria grew better than the wildtype bacteria, mainly presented on the maximal bio-mass at platform, suggesting that the VHb successfully worked on improving the growth of bacteria.

  Comparing the growth curve of the two VHb expressed bacteria we can see that the J23104 drived expressing of VHb can improve the growth of bacteria in a higher level.Allowing for that the expressing strength of J23104 is about twice stronger than J23110(characterized by iGEM2006_Berkeley), this result suggested that the more VHb expressed, the greater bacteria biomass would be gain at the platform, and that the expression strength of J23104 is not yet too strong to bring much burden to the bacteria. So in the future we are supposed to test several stronger promoter to find the best expression level of VHb.

  Next we compared the growth curve of intracellular expressing VHb bacteria and surface displaying VHb bacteria(fig.8).

  From this result we can see that with the same expressing strength, the growth rate of INP-VHb transformed bacteria is higher than VHb intracellular expressing bacteria, which fitted our modeling result(fig.9 modeling on the exponential phase, K1 represents the gathering rate of oxygen by VHb), while the maximal bio-mass at platform is lower and had no difference with the control group. So maybe the surface display of VHb would mainly affect the growth rate of bacteria but do little to the biomass for it’s dissociation from the respiratory chain.

  However, this result is still too weak to support this conclusion, so in the future we will adjust the expression level of surface displaying VHb to see their effects on the growth curve both in the growth rate and the maximal biomass. Also, we will apply the intimin and autotransporter to this system to test the effect of VHb on the growth ability of bacteria under different surface display system.

  Moreover, the difference between the growth curves was not very significant, and we thought maybe the normal condition contained enough oxygen so that the bacteria can grow well themselves. So in the future, we are supposed to optimize the experimental condition such as creating a low-oxygen content environment and so on.