Part:BBa_K3033023
BBa_K3033023: NSP4_Tyrosinase Secretable tyrosinase system
The second and very important feature for the whole Self-Activating Nanoparticle E.coli organism (SANCE). This feature is necessary for the activation of our first feature via the secretion of tyrosinase into the supernatant. The general overview of the whole feature is shown in Figure 1 below.
Tyrosinase is an oxidase which we would be using to synthesize L-DOPA on the cell surface of our engineered organism. We are using this enzyme to convert our expressed L-tyrosine on the capturing domains of the surface capture protein to L-DOPA. The chemical is as shown as in Figure 6a below. We have used a tyrosinase coding sequence (BBa_K3033013) which was derived from the tyrosinase producing bacteria called Bacillus megaterium strain M36.
Our second feature is a tyrosinase secretion system. This is a necessary system to have for the whole idea of our project due to the inability for L-Dopa to be synthesized naturally by E.coli. Therefore, our E.coli would be secreting an active form of tyrosinase into the supernatant where our organism would be located in, so that it would have contact with our capturing domains and convert tyrosine to L-Dopa. We plan to use the NSP4 signal sequence to carry our protein of interest to the extracellular environment. [2.2] Through the Signal Recognition Particle mediated sec-dependent pathway A as shown in Figure 6b above our protein would be secreted into the outside of the cell.
Protein expression tests
We cultured NSP4-tyrosinase transformed bacteria, saved some volume of the culture as uninduced bacteria sample and induced the rest volume of bacteria culture with 0.2% arabinose for the next 6~8 hours. Next, we centrifuged both induced and uninduced samples, we saved both pellet and supernatant for target protein expression check using western blotting. We lysed the cells using a lysis buffer. However, in this first western blotting result as shown in Figure 2, we were only able to clearly show that our target protein is expressed in the pellet sample. Tyrosinase which has the size of 34kDa has a strong detection signal compared to the uninduced samples. Although we got the right protein size, we want our protein to be secreted onto the supernatant. A possible explanation for this was that our protein concentration was not high enough in the supernatant sample thus we couldn’t see a signal for it.
The results of Figure 2 shows that there is not enough tyrosinase in the supernatant therefore we have performed Nickel pull-down. Figure 3 above shows our results after pulling down the supernatant samples. It makes sense that our target protein is not detectable in either before (C sample) or after pull-down (D sample) in the supernatant from bacteria transformed with vector without tyrosinase fragment insert after centrifugation. By concentrating our protein of interest from the supernatant using pull-down assay (B sample), we were able to see, as shown in Figure 3, the band of our target protein Tyrosinase, which is 34kDa in size. Indicating that it was pulled-down from the supernatant of induced samples while our target protein bands don’t show in the supernatant from bacteria transformed with tyrosinase gene without pull down. This result indicates that our protein is secreted in the extracellular space due to the NSP4-tyrosinase inserted in the pBAD24 vector backbone as it does not express in the bacteria sample which is only transformed with pBAD24 vector backbone without the insert.
Functional Tests
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In order to investigate the best condition for activating the tyrosinase secretion system, we induced the E.coli with a series of concentrations of arabinose ranging from 0 to 133000μM as well as two different incubation times: 4 and 8 hours under each concentration. In Figure 4, the tyrosinase activity increases while being induced by a higher concentration of arabinose, it reaches the highest tyrosinase activity when the concentration of arabinose added is the highest. However, longer incubation time would not contribute to a higher tyrosinase activity as the activity obviously does not increase much with 8 hour incubation even with very high arabinose concentration. Based on this functional test result, we can deem 133000μM arabinose induction with 4 hours incubation time with the induction chemical arabinose to be the best condition to collect the supernatant of bacteria culture and be used for further tests. With a higher concentration of arabinose induction, the expression of tyrosinase will increase in an appropriate incubation time length and would be able to show better enzyme function. The results that we have obtained shows that we are successful in secreting a functional tyrosinase enzyme out of our bacteria.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1511
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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