Team:ManhattanCol Bronx/Experiments

Experimental

This section will detail our experimental procedures and protocols.

Filler space

gBlock and Vector Ligations

Enzymatic Digests:

Our gBlock designs are described on the design page. Here we will detail the digest and ligation of those blocks into our vectors.Each of our blocks was design to use the prefix EcoRI and suffix PstI sites for insertion into both the pCDF-duet and pSB1C3 vectors.

Digestion of the gBlocks and plasmids were performed as follows:

• 100-200 ng of gBlock DNA was digest in a total volume of 10-15 µl. Our digested DNA concentrations were in the range of 5 - 10 ng/µl (final concentration).
• NEB buffer 2 was used at a 1X final concentration.
• 0.5 µl of EcoRI (NEB) and 0.5 µl of PstI (NEB) was used in each reaction.
• The reactions were incubated at 37C for 1 hr and then the enzymes were heat inactivated at 80C for 20 min.

When pCDF-duet was digest we used ~1000 ng of DNA and then separated the linearized backbone using a 0.8% agarose gel. The DNA was removed from the gel and purified using the Thermo Scientific GeneJET Gel Extraction Kit.

We followed the iGEM protocol for digesting the linearized backbone of pSB1C3 prior to ligations.

Ligations:

Ligation of our gBlocks into the vector backbone was performed as follows:

• 25 - 50 ng of digested vector DNA was used for each ligation.
• A 3-fold molar amount of digested insert (gBlocks) was used. Negative controls contained water in place of the insert.
• T4 DNA ligase buffer was used at a 1X final concentration.
• Water was added to adjust the final volume to 10 µl.
• The reaction was incubated at 16C for 30 min and then heat inactivated at 80C for 20 min.
• Transforms were performed using 2 ul of the ligation reaction.

Transformations:

Ligation transformations were performed with DH5alpha cells and performed as follows:

• 2 ul of ligation reaction was added to 5 ul of chemically competent cells and incubated on ice for 30 min.
• The cells were then heat shocked at 42C for 90 s and then directly chilled for 2 min.
• 500 ul of SOC was added and then the cells were incubated at 37C for 1 hr.
• The plates were incubated at 37C overnight.

Transformation verification:

Ligation verifications were performed by digest reaction of plasmid DNA that was isolated from cell colonies that grew in the presence of the appropriate antibiotics. The digestion protocol was identical to that stated above and then the DNA fragment products were then separated on a 1% agarose gel. Digestions that produced 2 fragments (the size of vector and insert) verified that the ligation worked in those clones and the DNA was then sequenced. The cells that harbored the correct ligation products were then glycerol stocked and stored at -80C. These results are outlined on our results page.

GOx Purification and Enzymatic Analysis

GOx Expression and Lysis:

Our expression system was performed with the vector pCDF-duet. The pCDF plasmid utilizes a T7lac promoter that is IPTG inducible. This vector requires an E. coli expression host (DE3) containing a chromosomal copy of the gene for T7 RNA polymerase. We transformed BL21(DE3) cells with DNA from successful ligation reactions. The transformation protocol was the same as detailed above. Cells carrying the appropriate vector were then grow in LB medium, at 37C, supplemented with spectinomycin. From an overnight starter culture we inoculated a 500 ml culture for protein expression and purification:

• Cells were allowed to grow to an OD600 = 0.8-1.0.
• IPTG was then added to a final concentration of ~ 400 uM.
• Cells were then allowed to express protein for ~ 16 h. Cell aliquots were taken for protein production analysis at several time points.
• Lysozyme (~10 mg) was added to the cell solution, mixed well and then incubated on ice for 30 min.
• The lysis was then vortexed and clarified at high speed centrifugation (~18K rpm, 20 min).
• The supernatant was collected and used as the load sample for the Ni²⁺-NTA column.

GOx Expression Western Blot Test:

Our GOx genes were designed with a 6x HIS-tag and we utilized this tag as a means of detecting the protein through Western blot analysis. The fractions that were taken at time points following IPTG induction were lysed by boiling 100 ul of cells (pelleted) in 100 ul SDS running buffer. The whole cell lysate was analyzed by SDS-PAGE electrophoresis on a 15% gel. Separated proteins were blotted to PVDF membrane in Towbin buffer (25 mM Tris, 192 mM glycine, 20% (v/v) methanol (pH 8.3)). The blotted proteins were decorated with anti-HIS primary antibody and then secondary antibody conjugated with HRP. We then visualized our proteins using ECL substrate.

GOx purification:

Our GOx expression lysates were passed over a 500 ul bed volume of BioRad, Profinity™ IMAC Resin, Ni-charged (#1560131), pre-equilibrated with PB buffer. The flow through was collected and the resin was washed with excess PB buffer. GOx was eluted using PB buffer supplemented with 250 uM imidazole. Elutions were performed as 5x 1 ml fractions. Each fraction was tested for presence of protein as well as the activity of GOx.

GOx Enzymatic Activity Assay:

GOx catalyzes the oxidation of D-glucose and produces hydrogen peroxide (H₂O₂). We took advantage of the Invitrogen Molecular Probes Amplex® Red Glucose/Glucose Oxidase Assay Kit (A22189) which allows for a one-step detection of glucose oxidase activity by coupling the production of H₂O₂ to the activity of horseradish peroxidase (HRP). H₂O₂ reacts with Amplex® Red (a colorless molecule), in the presence of HRP, to yield resorufin (red-fluorescent product). This colorimetric assay directly couples the activity of glucose oxidase to the production of resorufin.

We used the Amplex Red kit according to the manufactures protocol. To 50 ul of the assay reaction mixture (PB buffer, HRP, glucose, and Amplex Red) we added 50 ul of our elution fractions. The kit provided Wt GOx which was used as the positive control (0.1 U/ml final working concentration). Our negative control was elution buffer only.

We observe GOx activity from each of our tested constructs. The results are outlined on the results page.

nANODE Synthesis

To create our nanowires, we used polycarbonate filter membranes (templates) with track etched pores with a diameter of 200 nm. These templates were then sonicated in a bath sonicator in pure ethanol for approximately 10 seconds to fill the pores with ethanol. Following the sonication step, the template is then mounted into a U-Tube reactor to separate the two half-cell compartments. A picture of the assembled U-Tube is shown below.

A 0.005 M solution of HAuCl₄ is used to fill one side of the U-Tube apparatus. Simultaneously a 0.005 M solution of NaBH₄ is used to fill the other side. It is critical to fill both sides at the same time and same rate to ensure that the delicate template is not broken. Once filled, the U-Tube is left to react for a particular amount of time. The specific amount of time depends on several things, concentration of reactants, pore size, and temperature.

After allowing the reaction to occur, the solutions are carefully poured out of the U-Tube, the Au solution is saved for later use, while the NaBH₄ solution is discarded. The U-tube apparatus is dismantled and the template is carefully removed and dried with a Kim-wipe. A picture of the polycarbonate template after reaction is shown below. The gold colored portion is where the reaction occurred within the pores. We are unsure at this point why the reaction did not occur evenly over the entire template, but the entire area should have a gold color.

To isolate the nanowires from the template for imaging in a Scanning Electron Microscope, first the excess Gold is removed from the surface of the template by grinding with a grinding stone. The template is then placed into a centrifuge tube and dissolved using Methylene Chloride. The AuNWs are then removed by centrifugation at 4000 RPM for 7 minutes, and the supernate is discarded. This process is repeated 3 more times to ensure all of the polycarbonate has been removed. At this point, the AuNWs are ready for characterization with an SEM or to be used in electrochemical experiments. We have not had a chance to run any electrochemical experiments yet, unfortunately.