Part:BBa_K3589202
L1 – Wildtype marLac + HA (Phytobrick)
This composite part contains the PAR-promoter (BBa_K3002027) in combination with the RPL23-Terminator (BBa_K3002006), a 3xHA-tag (BBa_K3002017) and the coding sequence of the wildtype laccase from an uncultured marine bacteria (BBa_K3589109) plus the MoClo connectors for positions B2-B3 ( BBa_K3002303), B4-B5 ( BBa_K3002304) and B5-B6 (BBa_K3002305).
Summary of the Results from Team Kaiserslautern 2020 for part BBa_K3589202
- Intracellular expression could be achieved in C. reinhardtii when combined with a spectinomycin resistance (specR). See part BBa_K3589208
- Intracellular protein showed no activity in ABTS-assay
Cytosolic Expression:
Proof of Expression:
We first designed constructs without any secretion signal and transformed them into C. reinhardtii to make sure that the green alga is capable of producing both enzymes. We chose the strain UVM4 1 as a recipient.
a) Level 2 MoClo construct containing the coding sequence for marLac and a 3xHA-tag for detection as well as the pAR-promotor and the tRPL23-terminator. SpecR is a spectinomycin resistance cassette built from the level 0 parts PPSAD, aadA and PSADter introduced by team TU Kaiserslautern 2019 as part of the Kaiser Collection.
b) Immunoblot of 12 randomly picked spectinomycin-resistant colonies transformed with construct a). They were inoculated in TAP medium under mixotrophic conditions. Total protein samples were analyzed via SDS page and immunoblotting using anti-HA antibody. 2 µg of chlorophyll were loaded onto the gel. Expression of marLac (~ 60kDa) is visible in transformant 12. The recipient strain served as a negative control, a 3xHA-tagged protein as a positive control.
Measuring Activity of the Intracellular Laccases:
To measure the activity of the expressed cytosolic laccases, we performed an ABTS-assay. The assay is based on the change in color which appears when ABTS is oxidized to its corresponding radical cation. We performed this assay in a 96-well plate. A commercially available laccase from the fungus Trametes versicolor served as a positive control, supernatant of the lysate from the recipient strain as a negative control. First, we tried to lyse the cells by vortexing them with glass beads. The supernatant was then used for the assay.
Fig. 2: Activity assay for the cytosolically expressed enzyme marLac.
a) Cytosolic level 2 constructs of and marLac. b) ABTS activity assay performed in a 96-well plate. Absorption was measured at 415 nm. The error bars represent the standard deviation of four independent replicates for each sample. The concentration in the parentheses represents the final concentration of the whole protein in the 96-well plate. For the positive control, 75 µg of commercially available Trametes versicolor laccase was added to the 375 µg/ml of whole cell protein in 200 µl of lysate from the recipient strain. Only one sample was measured for the negative control. Cells were lysed using glass beads. The assay was performed at pH 7. Normalization of the data was done by dividing each absorption value by the initially measured value for that replicate.
The assay allowed to monitor the activity of the commercially available laccase, as documented by the blue line in Figure 2 which indicates that the assay worked. Unfortunately, no activity could be detected for the recombinant protein marLac, when it is localized in the cytosol. We therefore tried different lysis methods, i.e. sonication and freeze and thaw to test if it impacted the amount of protein in the lysate or its activity. With the sonication method, we were able to load up to 500 µg of whole protein into the assay. At this concentration, chlorophyll hindered the measurement because of its own absorption at 415 nm, rendering the method useless. We therefore tried the freeze and thaw method to decrease the amount of chlorophyll in the lysate. The method was successful at reducing chlorophyll levels, giving us the ability to more whole protein into the assay. Unfortunately, we were not able to detect any activity with this method, either.
From these results, we concluded that either the concentration of the expressed laccases was too low or that the enzymes are not active when expressed in the cytosol. The immunoblot in Fig. 1 indicates that the expression level of marLac, is very low when compared to the positive control. It is also possible that, when the laccase is expressed in the cytosol, it is inactive because of the lack of essential post-translational modifications, which usually occur during the secretion process, i.e. gylcosylation. Laccases are usually heavily glycosylated. They typically contain 10 – 45 % carbohydrates, depending on their origin. The carbohydrates are believed to play a key role in the stability of the laccases and protect them from proteolysis and inactivation by radicals.2 Since the laccase is not produced by C. reinhardtii natively, it is also possible that chaperones required for the insertion of the copper ions are missing. To check the validity of one of these explanations, the assays could be repeated with concentrated enzymes, e.g., via affinity chromatography. When the results are still negative with a much higher laccase concentration, one can assume that the enzyme is in fact not active when expressed in Chlamydomonas.
Summary and Outlook
Summary
Over the course of iGEM, we were able to produce marLac in the cytosol of the green alga Chlamydomonas reinhardtii. We were not able to detect any activity for the enzyme using an ABTS assay.
Outlook
A large obstacle could be the activity of the enzymes. Expression of the laccases does not automatically mean that they show any activity. Prof. Dr. Antonio Pierik, an expert on iron-sulfur proteins explained to us that the incorporation of metallic ions into a protein is not a trivial process. As laccases are multi-copper enzymes, it could be that laccases expressed by C. reinhardtii would not be active at all. A screening for activity should therefore always follow a proof of expression.
References
(1) Neupert, J.; Karcher, D.; Bock, R. Generation of Chlamydomonas Strains That Efficiently Express Nuclear Transgenes. The Plant Journal 2009, 57 (6), 1140–1150. https://doi.org/10.1111/j.1365-313X.2008.03746.x.
(2) Agrawal, K.; Chaturvedi, V.; Verma, P. Fungal Laccase Discovered but yet Undiscovered. Bioresour. Bioprocess. 2018, 5 (1), 4. https://doi.org/10.1186/s40643-018-0190-z.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1312
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 249
Illegal PstI site found at 1312 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1312
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1312
Illegal NgoMIV site found at 1172
Illegal NgoMIV site found at 1891
Illegal NgoMIV site found at 2342
Illegal NgoMIV site found at 2895 - 1000COMPATIBLE WITH RFC[1000]
| None |