Part:BBa_K4229054
otsA under the regulation of T7 promotor and Lac operator
otsA under the regulation of T7 promotor and LacI operator. With this Biobrick and the Biobrick BBa_K4229055, we were able to produce Trehalose. The growth and trehalose production of the transformed E. coli BL21 were investigated in different media. We tested bacteria with the empty backbone pET-Duet and our plasmid pET-Duet-otsAB. We decided to use LB Medium with 2% Glocose as described. The bacteria were incubated at 37 degree and induced with 0.5 mM IPTG from an OD of 0.6. After that, a sample was taken every two hours. The last sample was taken after 12 hours. The samples were centrifuged, washed with 1 x PBS and resuspended in ddH2O. Lysis was carried out by sonication. The production assay was performed with the Trehalose Assay Kit from Megazyme. The protocol for the microplate assay procedure was followed.Using the kit, trehalose production can be detected as described. The result is shown in figure 1. The absorbance was plotted against time. The induced sample containing the plasmid pET-Duet-otsAB shows a maximum of 0.654 after 4 hours of incubation. After reaching the maximum, the absorbance subsequently decreases until after 12 hours it is only slightly above the other samples. The uninduced plasmid, as well as only the backbone (induced and uninduced) shows a significantly lower absorbance of about 0.32. This allowed us to show the production of trehalose. And thus, also confirm the functionality of our plasmid. We were able to detect a mass of 2 to 4 µg Trehalose.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 460
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |