Part:BBa_K3589210
Level 2 � spectinomycin resistance + cCA_marLac_SP20-HA-RGS-8His
This composite part contains a spectinomycin resistance (BBa_K3002102) and the wildtype laccase from an uncultured marine bacteria (BBa_K3589109) fused with a SP20-HA-RGS-8His-tag (BBa_K3589150) for an efficient secretion, easy detection via HA-antibody and purification via nickel-NTA-column.
This part was designed to allow synthesis due to a high GC-level.
Summary of the Results from Team Kaiserslautern 2020 for part BBa_K3589210
- No expression could be detected in C. reinhardtii.
Protein Secretion:
The Screening ProcessFor these experiments, we switched to the CC-4533 strain. TU Kaiserslautern’s 2019 iGEM team was able to produce good results with that strain for secreted proteins. It still has parts of its cell wall and is therefore more robust and better suited for its usage in a photobioreactor than the UVM4 strain.
First, we designed four different constructs: two for each laccase. Each construct contained a N-terminal secretion signal from the enzyme carbonic anhydrase (cCA) and an HA-8xHis tag for purification. The construct pAR-marLac -SP20-HA-RGS-8His additionally harbor a module consisting of a repeat of 20 serine and proline residues (SP20). This module was reported to strongly increase the secretion of proteins.1 TU Kaiserslautern’s 2019 iGEM team was able to increase the secretion of their proteins significantly when using this module. The other construct, pAR-marLac -HA-RGS-8His, was designed to verify the results of TU Kaiserslautern’s 2019 iGEM team regarding the positive effect of the SP20 portion on secretion efficiency.
Fig. 1: The laccase cannot be detected in the supernatant when fused to an N-terminal cCA secretion signal and a C-terminal (SP20)-HA-RGS-8His-tag.
a) and b) shows the constructs consisting of a spectinomycin-resistance cassette (SpecR), the pAR-promotor, the coding sequence for the respective laccase, and the tRPL23 terminator. c) and d) shows secreted proteins precipitated from the medium of twelve independent transformants generated with each of the four constructs. The respective constructs are shown above each immunoblot. 6 mL of supernatant were harvested, lyophilized and resuspended in SDS sample buffer. 15 µL of sample were loaded onto the gel and analyzed via immunoblotting using an HA-antibody. Secreted proteins precipitated from medium of the recipient CC-4533 strain served as a negative control, that of a transformant expressing cCA and SP20-HA-8His-tagged brazil nut 2S albumin as a positive control. Controls were treated like the samples.
Fig 3 shows that marLac could not be detected in the supernatant. We therefore lysed the cell pellets of the transformants to see if the proteins are produced but not secreted or if the cells do not produce the proteins at all.
Fig 2: The laccase could not be detected in the whole cell body when fused to an N-terminal cCA secretion signal and a C-terminal (SP20)-HA-RGS-8His-tag.
a) shows the construct .c) the proteins of the lysed cell pellets were analyzed by immunoblotting. Cell cultures were lysed by boiling them with SDS sample buffer. For the samples and controls, proteins corresponding to 2 µg of chlorophyll were loaded onto the gel and analyzed via immunoblotting using an HA-antibody. In panel b), lysate from a transformant expressing cCA and SP20-HA-8His-tagged brazil nut 2S albumin (see Fig. XYY) served as a positive control. The recipient CC-4533 strain served as a negative control in both cases.
In the course of the project we screened in total the supernatants of around 100 randomly picked spectinomycin-resistant colonies transformed with the two different constructs for marLac secretion. The laccase could not be detected in any case.
Since we were able show that marLac is expressed in the cytosol, we have three possible explanations for our inability to detect them in the supernatant: (i) the expression levels of the enzymes are so low that they cannot be detected via immunoblotting. (ii) the N-terminal cCA-tag does not efficiently drive secretion of the enzymes. (iii) Laccase is not compatible with the secretion pathway in C. reinhardtii, for example because chaperones required for the insertion of the copper ions are missing. The apoproteins lacking these copper ions might be rapidly degraded in the ER.
To test the validity of the first explanation, we designed new constructs with the secretion signals ARS and GLE, introduced by TU Kaiserslautern‘s 2019 iGEM team. We also tried going back to a 3xHA-tag instead of the one containing an 8xHis portion. Because expression could not be observed, neither with nor without the SP20-portion of the C-terminal tag, we reasoned that it is not causing the problem in expression. We therefore chose the SP20-3xHA tag. As TU Kaiserslautern’s 2019 iGEM team has shown, the SP20 repeat greatly enhanced the secretion of their extracellular proteins. That’s why we did not want to waive it. Unfortunately, we were not able to screen transformants with these new constructs because of time restrictions.
Meanwhile, we contacted Dr. Dietmar Schlosser, an expert on fungal laccases. Because laccases are multi-copper enzymes, he suggested that we increase the copper concentration in the medium to identify whether copper is a limiting factor for the expression of our laccases. The medium we used before was the revised TAP medium by Kropat et. al.2 The copper concentration in this medium is 2 µM. Dr. Schlosser suggested that we should try a concentration of around 20 µM copper. Back in the laboratory, we made a modified version of the TAP medium containing 20 µM copper.
Fig. 3: Immunoblots of the supernatant from 12 randomly picked spectinomycin-resistant colonies transformed with the respective constructs and grown in TAP medium with 20 µM copper.
a) shows the construct, c) shows secreted proteins precipitated form medium for each transformant. The respective constructs are shown above each immunoblot. 6 mL of supernatant were harvested, lyophilized and resuspended in SDS sample buffer. 15 µL of sample were loaded onto the gel and analyzed via immunoblotting using an HA-antibody. Secreted proteins precipitated from medium of the recipient CC-4533 strain served as a negative control, that of a transformant expressing cCA and SP20-HA-8His-tagged brazil nut 2S albumin as a positive control. Controls were treated equally to the samples. For the positive control, only 5 µL were loaded onto the gel.
With an increased copper concentration in the medium, no signal could be detected for the laccase. From this finding we concluded that copper was not a limiting factor for the expression of marLac.
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. The enzyme was not expressed when fused to a N-terminal cCA-secretion signal and a C-terminal (SP20)-HA-RGS-8His-tag. An increased copper concentration did not change these results.
Outlook
To test whether C. reinhardtii can express and secrete laccases, more experiments are required. Transformants of the designed constructs with different secretion signals, i.e. GLE and ARS and/or different tags, i.e. an SP20-3xHA-tag should be screened. Another 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 secreted 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) Ramos-Martinez, E. M.; Fimognari, L.; Sakuragi, Y. High-Yield Secretion of Recombinant Proteins from the Microalga Chlamydomonas Reinhardtii. Plant Biotechnol J 2017, 15 (9), 1214–1224. https://doi.org/10.1111/pbi.12710.
(2) Kropat, J.; Hong-Hermesdorf, A.; Casero, D.; Ent, P.; Castruita, M.; Pellegrini, M.; Merchant, S. S.; Malasarn, D. A Revised Mineral Nutrient Supplement Increases Biomass and Growth Rate in Chlamydomonas Reinhardtii: A Revised Mineral Nutrient Supplement for Chlamydomonas. The Plant Journal 2011, 66 (5), 770–780. https://doi.org/10.1111/j.1365-313X.2011.04537.x.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 2401
Illegal PstI site found at 3795 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 2401
Illegal NheI site found at 2665
Illegal PstI site found at 3795 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 2401
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 2401
Illegal PstI site found at 3795 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 2401
Illegal PstI site found at 3795
Illegal NgoMIV site found at 1401
Illegal NgoMIV site found at 1584
Illegal NgoMIV site found at 1694
Illegal NgoMIV site found at 3655
Illegal NgoMIV site found at 4374
Illegal NgoMIV site found at 4825
Illegal NgoMIV site found at 5471 - 1000COMPATIBLE WITH RFC[1000]
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