Part:BBa_K3589212

Level 2 � spectinomycin resistance + cCA_marLac_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 HA-RGS-8His-tag (BBa_K3589151) for easy detection via HA-antibody and purification via nickel-NTA-column.

Summary of the Results from Team Kaiserslautern 2020 for part BBa_K3589212

• No expression could be detected in C. reinhardtii.

Protein Secretion:

The Screening Process
For 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 (Spec^R), 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.

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.

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.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 2401
  Illegal PstI site found at 3795
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 2401
  Illegal NheI site found at 2665
  Illegal PstI site found at 3795
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 2401
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 2401
  Illegal PstI site found at 3795
  
• 25
  INCOMPATIBLE 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 5345
  
• 1000
  COMPATIBLE WITH RFC[1000]