Team:SCAU-China/Engineering
For MESEG project, we are planning to construct a fusion protein consisted of an ATG8 interacting motif (AIM), a MT that scavenge heavy metal, and a fluorescent protein for tracing. In this AIM and MT combination, the MT could be alternative according to its targeting heavy metal, which can be flexibly modified/substituted so that indeed expanding the target spectrum. We reasoned that the fusion protein, when expressed in Chlamydomonas, would specifically bind heavy metal via MT and, through the interaction between the AIM and ATG8, deliver the fusion protein–heavy metal complex to the vacuole, thus enhance its phytoremediation capability. So far, we went through an iteration of the engineering design cycle required by iGEM and demonstrated that our project was partially succussed.
Video 1: The demonstration video of MESEG Project
As a proof of concept of MESEG, we first chose yeast as a model to test the phytoremediation function of our constructs.
First Try of Wet Lab
We first used ATG19, a well-known yeast receptor as the starting material for MESEG construction. In yeast, the receptor ATG19 is required for the vacuolar deposition of Ape1, a vacuolar hydrolase. It binds Ape1 through its Ams1 binding domain (ABD) in the middle and binds ATG8 through ARR domain at its C-terminal (Yamasaki and Noda, 2017). It also can self-oligomerize through its coiled-coil (CC) region. Therefore, we kept the ARR domain and CC region, but replaced the ABD with SpMTL, a Cd binding protein derived from Sedum plumbizincicola and tagged a mCherry florescence protein at its N-terminal.
When the construct mCh-CC-SpMTL-ARR was expressed in the Cd-sensitive mutant Δycf1, it only slightly increased its Cd tolerance as compared with the construct with SpMTL (mCh-CC-ARR), but much weaker than the wild-type strain BY4741 (Figure 1). When the ARR domain is removed, the expression of mCh-CC-SpMTL conferred Δycf1 mutant better Cd tolerance，indicating that the Cd binding of SpMTL might be interfered by the up and/or downstream domains.
Figure 1. First generation of MESEG protein mCh-CC-SpMTL-ARR did not confer yeast Cd tolerance
The wild-type yeast strain BY4741 or Cd-sensitive mutant Δycf1 were transformed with mCh-CC-ARR, mCh-CC-SpMTL-ARR or mCh-CC-SpMTL and grown on SD plates with indicated concentrations of CdCl2 for 3 d.
Moreover, using confocal laser scanning microscopy, we found that the protein mCh-CC-SpMTL-ARR is recruited to the pre-autophagosomal structure (PAS) regardless of nitrogen status (Figure 2), which is is similar to that of autophagic marker ATG8, implying that that this protein might might behave like cargo receptor ATG19 and is transported to the vacuole via autophagy route.
Figure 2. The subcellular localizations of fusion protein mCh-CC-SpMTL-ARR
The Δycf1 mutant were transformed with pYES2 vectors expressing mCh-CC-SpMTL-ARR fusion proteins, and grown under nitrogen rich(+N) or nitrogen deficiency conditions (-N) for 12 hr before Confocal microscopy observation.
Dry Lab：Simulation Analysis of MESEG Constructs
We then performed molecular simulating analysis of fusion protein mCh-CC-SpMTL-ARR using trRosetta software and tried to separate each domain with various types of linkers. As shown in Figure 3, in the absence of linker or presence of flexible linker, the MT within the fusion protein is entangled with other structures, and its binding capability is compromised. However, after adding rigid linker, each domain can be separated effectively, indicating that rigid linker could be used to improve the binding capability of MTs (Fig.4). We also found that the entanglement may be caused by the formation of many tight disulfide bonds between MT and CC or ARR, and the addition of the linker can alleviate this situation. For more information, please visit Model page.
Figure 3. Molecular simulation analysis of fusion protein mCh-CC-SpMTL-ARR with or without linker.
From left to right are fusion proteins without linker, with flexible linker or rigid linker. Red to purple patterns indicate regions/domains from N-terminal to C-terminal.
Figure 4. Schematic diagram of structure and function of fusion protein in linker mode
Second try of Wet Lab
We then redesigned our MESEG proteins by adding a rigid linker EAAAK and tested in yeast. As shown in Figure 5, expression of SpMTL-LEAAAK-CC-mCherry-ARR restore the Cd-tolerance of Δycf1 mutant, although was slightly weaker than wild type.
Figure 5. Expressing the fusion protein SpMTL-LEAAAK-CC-mCh-ARR restored the Cd tolerance of yeast mutant Δycf1.
The wild-type yeast strain BY4741 or Cd-sensitive mutant Δycf1 were transformed with EV (pYES2 empty vector), or pYES2 carrying SpMTL-LEAAAK-CC-mCh-ARR, and grown on SD plates with indicated concentrations of CdCl2 for 3 d.
Figure 6. The subcellular localizations of fusion proteins SpMTL-mCherry and SpMTL-LEAAAK-CC-mCh-ARR
The Δycf1 mutant were transformed with vectors expressing SpMTL-mCherry or SpMTL-LEAAAK-CC-mCh-ARR fusion proteins and grown under nitrogen deficiency conditions for 12 hr before Confocal microscopy observation. V, vacuole.
The above results demonstrated that the phytoremediation capability of our MESEG protein SpMTL-LEAAAK-CC-mCh-ARR improved significantly according to molecular simulation analysis. It made us successfully complete a cycle of design → build → test → learning → design, which is the requirement of silver medal award.
We further optimized the linker and found that a linker LRRRF gave the best Cd tolerance performance (Fig.7).We also find the reason about the super power of LRRRF added fusion protein. Please refer to Model for details.
Figure 7. Addition of LRRRF linker peptide conferred the best Cd tolerance.
The wild-type yeast strain BY4741 or Cd-sensitive mutant Δycf1 were transformed with EV (pYES2 empty vector), or pYES2 carrying mCherry, SpMTL, SpMTL-mCh, SpMTL-LEAAAK--CC-mCh-ARR or SpMTL-LLRRRF-CC-mCh-ARR, and grown on SD plates with indicated concentrations of CdCl2 for 3 d.
MESEG Proteins Expression in Algae
We also tried to express MESEG proteins in Chlamydomonas. Unfortunately, our transformed Chlamydomonas clones were always contaminated by fungi during clone screening (Figure 8). We have carried out Chlamydomonas transformation experiments for three times but could not obtain fungi-free positive clones. Until recently, with the help of Professor Keke Yi (Institute of agricultural resources and regional planning, Chinese Academy of Agricultural Sciences), we have successfully obtained contamination-free algae clones (Figure 9). At present, we are conducting follow-up experiments.
Figure 8: Fungi contaminated Chlamydomonas clones.
Figure 9. Identificaiton of Chlamydomonas clones by using PCR.
CK+ indicates vector containing target gene as positive control; CK- is empty vector without target gene. S1~S7 are samples.
References
- Yamasaki, A., and Noda, N.N. (2017). Structural Biology of the Cvt Pathway. J MOL BIOL 429, 531-542.
