gBlock and vector cloning
Following each digest and ligation, single clones were selected and grown in media supplemented with the appropriate antibiotic. The DNA from each clone was then isolated and test digested with the same enzymes used prior to ligation. DNA that yielded the correct insert and vector DNA fragments were then retained, sequenced, and stored for further use. We used EcoRI and PstI sites for the creation of our vectors and therefore we used the same enzymes to test the ligation products for the proper insertions.
Figure 1. gBlocks of GOx Wt (part 2238000) and GOx-4mut (part 2238001) were cloned into the vector pCDF. Clones that conferred resistance to spectinomycin were cultured and then the DNA isolated and test digested with EcoRI and PstI. In our first cloning attempt we were only successful with the GOx-4mut and not the Wt. We sequenced pCDF-GOx-4mut clone 1 and used this vector for the remainder of our experiments.
Figure 2. We retried cloning GOx-Wt into pCDF-duet and picked new clones. The DNA was isolated and test digested with EcoRI and PstI. Our second attempt yielded positive clones. We sequenced pCDF-GOx-Wt clone 11 and used this vector for the remainder of our experiments.
Figure 3. We cloned gBlock GOx-cys (part 2238002) and GOx-4mut-cys (part 22380003) into the pCDF vector. We were unable to successfully acquire clones from our GOx-cys ligations but we did manage to succeed in making pCDF-GOx-4mut-cys. DNA from those ligations were test digested with EcoRI and PstI. Clone 1 of pCDF-GOx-4mut-cys was sequenced and used for the remainder of our experiments.
Figure 4. We then cloned all of our gBlocks GOx-cys into the pSB1C3 vector to create our biobricks. Again, we used EcoRI and PstI digests. Successful growing clones were then cultured, DNA isolated and test digested. EcoRI and PstI were expected to give two fragments of nearly the same size, so we chose to test digest with BspHI. Successful ligations would yield digests that gave two bands at approximately 3000 and 1000 bp. In our first attempt we successfully cloned GOx-cys, GOx-4mut, and GOx-4mut-cys into pSB1C3.
Figure 5. We retried our ligation to make pSB1C3-GOx Wt. Again, we used EcoRI and PstI digests to create the vector but test digested with BspHI. 2 of the clones selected displayed the correct fragments following digestion. Clone 1 was sequenced and sent to iGEM.
GOx Test Expressions and Colorimetric Activity Assay
Each of our pCDF-GOx constructs was transformed into BL21(DE3) cells for IPTG inducible expression. Cells were collected at several time points following induction to test for the presence of GOx protein. Whole cell lysates were separated by SDS-PAGE and then blotted. Our constructs express proteins that contain 6x HIS tags and we decorated our blots against that HIS tag.
Figure 6. Western blot analysis of test expressions of GOx Wt, 4mut, and 4mut-cys. In the presence of IPTG there is protein present.
We could clearly demonstrate that protein GOx protein was produced in the presence of IPTG. We choose to express protein overnight in 500 ml of cells and then isolate GOx from those expressions. Using the BioRad Ni2+-NTA system described in the experimental we isolated each of our GOx proteins in 5, 1 ml elutions. Each of those elutions were then tested for GOx activity using the Amplex Red activity kit described in the experimental. Elutions that contained active GOx convert Amplex Red into a visible red dye in this colorimetric assay.
Figure 7. Amplex Red assay. GOx positive control is performed with GOx from the Amplex Red kit. The negative elution buffer row are elutions from an expression performed from an empty pCDF vector. The relative colorimetric changes are after 30 min reaction time. There is activity in each of our isolations, however at a slower activity rate than the given Wt control.
Figure 8. The Amplex Red assay was allowed to react for 3 hours. The increased time allows for a clearer representation of the colorimetric change due to the enzyme activity.
Continuing Work
Unfortunately, by the time of the wiki freeze we were still not at our intended goal. We are working on quantifying our GOx protein and using the Amplex Red kit to determine its actual activity. We feel that our gold anode is ready and we are in the process of setting up our fuel cell. We are working to have that data between the wiki freeze and the Jamboree presentation.
