We performed a new round of phenotyping assay for pDawn-RBSNNN-X174E-rrnB T1 (BBa_K3740044). First, the monoclonal transformants of E. coli TOP10 with pDawn-RBSNNN-X174E-rrnB T1 were picked and grown on two parallel new LB plates, with one cultured in the dark and the other under blue light respectively to screen for correct builds. As shown in Figure 1, pSEVA331-pDawn-RBSNNN-X174E-rrnB T1 had a total of 10 strains which survived in the dark but failed to grow under blue light. This suggests that the blue light induced lysis system can function as intended in E. coli TOP10.
From the above successful recombinant candidates (red squares in Figure 1), two bacterial strains were picked and grown in two parallel tubes of LB medium respectively, with one tube in the dark and the other tube under blue light to track. The OD600 values of the two strains cultured under the two different illumination conditions were measured at hourly intervals. As shown in Figure 2, both strains grew normally in the dark. For the #1 strain, the OD600 value increased during the first four hours of blue light illumination and then declined significantly. This delay in lysis indicates it takes time for the lysis protein X174E to accumulate to lethal concentration under blue light. That is to say, the blue light sensitive lysis system of this strain is not efficient enough. For the #11 strain, the OD600 value remained low during the first five hours of blue light illumination indicating the effectiveness of the system, but then increased dramatically implying that loss-of-function mutations could occurred in these genes or in the host genome and that the favorable mutant overtakes the parental population. Therefore, the blue light sensitive lysis system of this strain is not evolutionarily stable.
To enhance glutathione production in G. hansenii ATCC53582 , we constructed two expression systems of the bifunctional glutathione synthetase GshF on the basis of two pSEVA331 plasmid derivates, with one harboring the CmR gene while the other one carrying the AmpR gene. The resultant two plasmids of pSEVA331-gshF were introduced into G. hansenii by electroporation separately. As shown in Figure 3, the two recombinant strains with different antibiotic resistance have significantly different levels of glutathione. One possible reason is that resistance gene influences the expression of GshF. It is also likely that the antibiotic molecules affect the activity of GshF. We hope that future iGEMers will make different attempts on the resistance genes in their vectors to optimize their system.
To generate stable recombinant bacterial strain, we were supposed to use chloramphenicol at a concentration of 148 ng/mL to cultivate G. hansenii containing the plasmid pSEVA331 (CmR). However, we mistakenly used this antibiotic at a lower concentration of 37 ng/mL, and we unexpectedly found that the bacteria grew faster in this "wrong" LB medium. To quantify this difference, we measure the OD600 values of the strain in LB medium with different concentrations of chloramphenicol (37 or 148 ng/mL) and plotted the growth curves (Figure 4). If future iGEMers have problems with bacterial growth, we recommend them to change the concentration of antibiotics in medium to obtain the optimal amount of bacterial biomass.
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