We obtained E. coli ME8077 strain (minA minB thr-1 leuB6 azi lacY1 tsx ara-13 gal-6 malA1 thi-1 xyl-7 rpsL mtl-2 tonA2 supE44) from NBRP (NIG, Japan). Since ME8077 is a knockout of minB locus (which includes minCDE gene cluster), it produces minicells. We incubated ME8077 strain in LB broth overnight, and observed the morphology of cells under confocal microscope to confirm the production of spherical minicell and long rod shaped cell, which are both result of a uneven distribution of genetic material during cell division. Furthermore, we measured and plotted the length of cells from the confocal photomicrography using ImageJ.
Figure 1. (a) Minicells observed from E. coli ME8077 (b) Rod shaped cells that produce minicells
Figure 2. Cell length distribution of E. coli ME8077 strain
After synthesizing the Insulin production cassette flanked by biobrick prefix and suffix, we first digested the part and pSB1C3 vector with EcoR1 and Pst1 and then ligated the two parts. The resulting plasmid was transformed into E.Coli DH5-α.
Figure 3. E. coli DH5 alpha transformed with ligated plasmid
We runned gel with the miniprepped DNA but by EcoR1, which should be about 2.5k long. We found out that only one of the three plasmids, were correctly transformed, which was the part with constitutive promoter J23100.
Figure 4. 1.2% Agarose Gel electrophoresis result of pSB1C3 cut with EcoR1
In order to detect Insulin production, we conducted SDS-page gel using the DH5 alpha tranformed with the J23100-CPP-Insulin in pSB1c3 grown in LB medium overnight.
Figure 5. 10% SDS-Page Gel showing expression of protein
In future study, we are planning to transform the ME8077 strain using the prepared DNA.
