Maganese Peroxidase

Manganese peroxidase is an enzyme from the fungus, Phanerochaete chrysosporium. In this fungus, commonly known as white rot fungus, this enzyme aids in lignin degradation. Its enzymatic activity has been found to be optimal near a pH of 4.5 and at a temperature of 32C.

Syringaldazine Oxidation Assay

Syringaldazine (4-Hydroxy-3,5-dimethoxybenzaldehyde azine) can be used as an indicator of oxidative activity for laccases and peroxidases. The general conditions outlined by the protocol given on the Sigma Aldrich website were used in order to preliminarily assess if the mnP coding sequence encoded by the BBa_K1357001 on the compound BioBrick BBa_ K1357006 was producing MnP protein. The operating pH for this assay is 6.5 and hence fell far outside the pH optimum for the MnP protein. Even so, BioBrick BBa_ K1357006 showed an observable difference in oxidative activity (see Figure 1 below).

Nylon Degradation Assay

In order to assess if BBa_ K1357006 would be able to be used as a means of degrading nylon 6,6 micro and macro plastics, our team developed a nylon degradation assay. Samples wit approximate diameters of 0.5mm were submersed in 3 mL of growth medium with BL-21 E. coli cells with BBa_ K1357006 and incubated in a shaker at 32℃ and 160 rpm for 1 to 3 days. The growth medium was supplemented with manganese sulphate as well as a buffered solution comprised of sodium acetate and acetic acid so as to lower and maintain the pH of the mixture to 4.5 for the duration of the experiment. The samples were massed before and after incubation, but no noticeable change in mass was observed. Scanning electron microscopy (SEM) was used in order to visualize the surfaces of treated and untreated samples of Nylon 6,6.

The results from the SEM imaging showed physical deformations on the surfaces of samples treated with BL-21 E. coli cells with BBa_ K1357006 that were not evident in any of the untreated samples or controls that were shaken in media without cells present. These physical deformations were not consistent in their appearances. Further experimentation before the 2014 jamboree is intended to explore the effects of long term exposure, to improve the consistency of the effect, establish a quantitative means of measuring the effect of our modified bacteria.

Overview:

NylC is a nylon hydrolase derived from Flavobacterium sp. K172 that digests the internal amide bonds in nylon 6. It hydrolyzes the oligomer form of nylon 6 and liberates primary amino groups as the oligomer is broken down into monomer ¹. These free amino groups can be detected by trinitrobenzene sulfonic acid, which can react with primary amino groups to form orange-colored products whose absorbance is at 335-345nm².

The nylC Biobrick (BBa_K1357007) is introduced into E. coli BL-21 and the bacteria are cultured for approximately 20 hours. After centrifugation, the cell pellets are lysed by lysozyme and the resultant lysate which contains nylC enzyme is used to degrade nylon 6 samples³. Mass difference of nylon samples before and after experiment are determined and the nylon is imaged using scanning electron microscope (SEM). In addition, trinitrobenzene sulfonic acid is used to test the production of primary amino groups in solution.

Buffer Preparation:

1. Since nylC has its optimal activity at pH between 7.2-7.3, both the monobasic and dibasic of potassium phosphate (KH₂PO₄ and K₂HPO₄) are used to prepare buffer A¹. The concentration of stock solution of buffer A is made to be 100mM potassium phosphate (KH₂PO₄ and K₂HPO₄) and 10% glycerol. The pH used in experiment was 7.23.
2. Lysis buffer is required to disrupt bacterial cells. 1M Tris-HCl (pH=7.5), 1M DTT, 100mM PMSF are prepared as stock solution beforehand. The DTT and PMSF are aliquoted and stored in -20 degree freezer. The working solution of lysis buffer contains 50mM PMSF, 5% glycerol and 1mM DTT.

Procedure:

1. Culture BL-21 (DE3) E. coli containing nylC Biobrick in 200-300mL liquid LB (with 20 μg/mL chloramphenicol) for 20 hours in 37 degree incubator.
2. After centrifugation, resuspend every 1 gram of cell pellet with 1 mL of chilled lysis buffer. Then add 10μL PMSF (100mM) for every 1 mL of cell suspension.
3. Add lysosyme into the suspension to reach the final concentration 1mg/mL and incubate the cell suspension at 4 degrees Celsius for 4 hours.
4. Add 5 μL MgCl2 (1M) and 1μL DNase (1mg/1mL) for every 1mL of cell suspension and incubate the solution at 4 degrees Celsius for 30 mins.
5. Remove cell debris by ultracentrifugation at 4 degrees Celsius for 30 mins. (45000 rpm wih a 45Ti rotor, Beckman)
6. After centrifugation, recover the supernatant (lysate) and evenly distribute into three ependorf tubes. Discard the cell pellet. Add equivalent volume of Milli-Q water into two of the three tubes so the concentration of lysate halves. For the remaining one tube, add equivalent volume of buffer A to halve the concentration of lysate. Set up another eppendorf tube and add Milli-Q water into it so that it has same final volume of solution as the other three tubes.
7. Cut thin nylon 6 ropes into three small pieces and weigh each on mass balance. Carefully use a tweezer to immerse each of the three nylon pieces into three tubes containing Milli-Q water (control), lysate+Milli-Q water and lysate+buffer A. One of the four tubes does not contain a nylon 6 sample (control).
8. Incubate all four tubes in 30 degree incubator for 20 hours.
9. Take out nylon pieces, wash with Milli-Q water and let it dry overnight.
10. Use the same mass balance to weight the mass of three nylon piece again. Then image the three nylon samples using scanning electron microscope (SEM).
11. For the four reaction tubes, the concentration of primary amino groups in each is determined by trinitrobenzene sulfonic acid method ^2.

1. The cell lysis part of this procedure is adapted from website of European Molecular Biology Laboratory ^4.
2. We are still working on modifying the functional assay of nylC. The procedure presented above is the most recent one we used in the experiment.

Results:

There is no significant mass difference before and after treating nylon samples with lysate containing nylC. The SEM images show that there is no significant difference between the reaction group and control group. We propose two reasons to explain the results for both mass difference test and SEM imaging.

We suspect that due to inappropriate experimental procedures, such as leaving lysis samples at 4 degree fridge overnight and conducting experiment at room temperature instead of at 0-4 degree, activated protease degrades a significant amount of nylC enzyme. We will carefully conduct the experiment under low temperature and figure out a more effective way to inhibit protease activity.

We also suspect that the degradation effect of nylC activity is not reflected in both the mass difference test and SEM imaging. The mass is measured inaccurately because our mass balance had a maximum sensitivity of 1mg while the procedure required a resolution of 0.1 mg. In addition, the nylC may have its degradation effect on the two edges of the nylon strip instead on surface. We will perform trinitrobenzene sulfonic acid assay next to verify whether there is a positive change in the concentration of primary amino groups in solution, thus examine the degradation effect of nylC.

Chromoprotein Reporter Constructs

Building upon the work of the Uppsala iGEM team, we developed a series of reporter constructs based on the chromoprotein parts developed by Uppsala.

Uppsala made a wide array of chromogenic proteins available over the course of several years. They registered a vast array of parts, including coding sequences and coding sequences with attached ribosomal binding sites. We wanted to pick up where Uppsala left off and create a simple one step reporter BioBrick unit. We combined the generic, high-affinity ribosome binding site, BBa_B0034, a standard double-terminator, BBa_B0015, and the coding sequences for a purple (tsPurple), blue (amilCP), and red (mRFP) chromoprotein.

These BioBricks allow any user to verify the expression of a protein, by performing one step of BioBrick formation. All that is required is simply adding the devices we’ve created behind one’s protein of interest. These are some examples of what such a construct would look like this

Our Road to Gold!

Over these last few months we have worked our absolute hardest to compete at the international standard iGEM teams across the globe have set for themselves. Our path to a gold medal was all too literally paved with our blood, sweat and tears. Here's what we did: