Team:TU Kaiserslautern/Basic
Parts
The basic parts include coding sequences for the two laccases as well as two tags for C. reinhardtii. The laccase’s coding sequences are optimized for E. coli and C. reinhardtii respectively. All parts for C. reinhardtii are compatible with the MoClo-sytem. With these basic parts, we were able to build the foundation of our project.
Basic Parts
Thanks to last year’s TU Kaiserslautern iGEM team, we were able to use the Kaiser collection and build onto it. To help future iGEM teams use C. reinhardtii as a chassis, we registered two new tags for detection and two coding sequences for the laccases BaLac and marLac. These five parts have already been codon optimized for C. reinhardtii and were used during our project phase. For teams working with E. coli, we can offer the two new optimized coding sequences for the two laccases. These laccases show very interesting properties, such as pH stability in more neutral pH levels, and are a potential alternative for the often-used laccase from Trametes versicolor.
Best Basic Part
We can offer the coding sequence of the mutated Laccase from Botrytis aclada for E. coli . As an enzyme, it is able to oxidize certain micropollutants. In contrast to the often-used laccase of Trametes versicolor, it shows promising activity in a more neutral pH. Therefore, it is suitable for usage in scenarios such as water treatment plants.
Laccases for both Chassis
The wildtype B. aclada laccase has a high redox potential and shows a low inhibition by chloride. It demonstrates a clear pH optimum in acidic regions with a reasonable specific activity at pH 6.1We use a mutated version of B. aclada, namely with the point mutation L499F, which showed a better stability in more neutral pH levels and has a high redox potential. Thus, it is better suited for our goal to use the this enzyme in a bioreactor.2 The coding sequence of the used mutated B. aclada laccase (BaLac) for C. reinhardtii was split into two parts called BaLac part 1 and BaLac part 2.
As a second enzyme, we used the laccase-like enzyme marLac which was derived of a marine metagenomic library. It is thermostable and shows activity in neutral pH-levels. Furthermore, it is cold adapted and shows a high tolerance for organic solvents and salt.3
Table 1: Registered coding sequences for C. reinhardtii.
| Name | Type | Description | Designer | Length [bp] |
|---|---|---|---|---|
| BBa_K3589107 | Coding | Mutant BaLac for C. reinhardtii part 1 | Yannik Schermer | 1439 |
| BBa_K3589108 | Coding | Mutant BaLac for C. reinhardtii | Yannik Schermer | 2729 |
| BBa_K3589109 | Coding | Wildtype marLac for C. reinhardtii | Yannik Schermer | 1968 |
| BBa_K3589110 | Coding | Mutant BaLac for C. reinhardtii part 2 | Yannik Schermer | 1290 |
Table 2: Registered coding sequences for E. coli.
| Name | Type | Description | Designer | Length [bp] |
|---|---|---|---|---|
| BBa_K3589105 | Coding | Mutant BaLac for E. coli | Yannik Schermer | 1686 |
| BBa_K3589106 | Tag | Wildtype marLac for E. coli | Yannik Schermer | 1323 |
Tags for C. reinhardtii
During our project we used four different tags for C. reinhardtii. The tag used for cytosolic expression was a 3HA tag introduced by TU Kaiserslautern’s 2019 iGEM team.One was a combination of HA, 8His and RGS. The second one was a combination of SP20, HA, 8His and RGS. While the third tag is a combination of SP20 and 3HA.
The SP20 part of the tags contain 20 repeats of Proline and Serine. The proline is transformed into hydroxyproline which is then glycosylated. The glycosylation should increase secretion of proteins.4
The HA part of the tags allows detection via Western Blot with the primary antibody (anti-HA, mouse) and a secondary antibody (anti-mouse, rabbit) which has a conjugated horseradish peroxidase. This allows detection with chemiluminescence.
The 8His tag can be used for purification, because the histidine can bind to a Ni2+ NTA column. The eluted samples then can be separated by SDS-page and is detected by Coomassie-staining or HA-detection.
Table 3: Tags for C. reinhardtii submitted by iGEM TU Kaiserslautern 2020.
| Name | Type | Description | Designer | Length [bp] |
|---|---|---|---|---|
| BBa_K3589150 | Tag | SP20-HA-RGS-8His | Yannik Schermer | 195 |
| BBa_K3589151 | Tag | HA-RGS-8His | Yannik Schermer | 69 |
Regarding the used regulatory units, antibiotics resistance, secretion signals and scars, please check out these parts from iGEM TU Kaiserslautern 2019.
References
(1) Kittl, R.; Mueangtoom, K.; Gonaus, C.; Khazaneh, S. T.; Sygmund, C.; Haltrich, D.; Ludwig, R. A chloride tolerant laccase from the plant pathogen ascomycete Botrytis aclada expressed at high levels in Pichia pastoris. Journal of biotechnology 2012, 157 (2), 304–314. DOI: 10.1016/j.jbiotec.2011.11.021.(2) Scheiblbrandner, S.; Breslmayr, E.; Csarman, F.; Paukner, R.; Führer, J.; Herzog, P. L.; Shleev, S. V.; Osipov, E. M.; Tikhonova, T. V.; Popov, V. O.; Haltrich, D.; Ludwig, R.; Kittl, R. Evolving stability and pH-dependent activity of the high redox potential Botrytis aclada laccase for enzymatic fuel cells. Scientific reports 2017, 7 (1), 13688. DOI: 10.1038/s41598-017-13734-0.
(3) Yang, Q.; Zhang, M.; Zhang, M.; Wang, C.; Liu, Y.; Fan, X.; Li, H. Characterization of a Novel, Cold-Adapted, and Thermostable Laccase-Like Enzyme With High Tolerance for Organic Solvents and Salt and Potent Dye Decolorization Ability, Derived From a Marine Metagenomic Library. Frontiers in microbiology 2018, 9, 2998. DOI: 10.3389/fmicb.2018.02998.
(4) Ramos-Martinez, E. M.; Fimognari, L.; Sakuragi, Y. High-yield secretion of recombinant proteins from the microalga Chlamydomonas reinhardtii. Plant biotechnology journal 2017, 15 (9), 1214–1224. DOI: 10.1111/pbi.12710.
