Part:BBa_K3596056

BIOT with RBS mut4 and Pilin gene mut4A

BIOT is a basic design for the expression of Geobacter sulfurreducens's pilin-based electrically conductive protein nanowires (e-PNs)^[1]. The system includes a tac promoter which promotes the pilin gene(pilA) to express protein monomers; the monomers are later assembled into protein nanowires by the TypeIV pilin assembly system, which is constructed from the gene cluster at the downstream of the pilin gene(figure 1A). The e-PNs can generate electric potential with the presence of water molecules in the air, which can be further modified for other purposes.

Characterization

In order to improve the protein nanowire, we mutated the RBSs of the TypeIV pilin assembly system and the pilA gene to enhance the productivity and output voltage, respectively.

High-efficient production of protein nanowire for large scale application

First, Cost Matters!

The important reason why we choose E. coli to express nanowires is its abundant regulatory tools. It's easy to know that the Type IV system on the member could be one of the limiting processes in the nanowires assembly.

We then take the RBS optimization strategy to target the RBSs of the Type IV e-PNs assembly system. We expect this strategy can increase the number of type IV secretion machines on the membrane, thus optimizing the production of the e-PNs, then lowering the cost. As shown in the figure below, there are mainly three RBSs, which are RBS of hofB, hofM and ppdA. These three RBS sites have been reported in the literature that their sequence changes can optimize the expression of nanowires in E. coli

We originally planned to create a library for each RBS, then randomly combine them to find the optimum combination. So we used RBS calculator (Link) from Salis Lab, obtaining 9 more mutated sequences with various strengths for each. But, with ten sequences for each of the 3 RBSs, there're 1000 possibilities! Therefore we would need to spend 250 days on the purification process (4 purifications/Day). It is impossible in one-year iGEM Competition.

Figure 1: Building RBS library to Improve the production of BIOT.

Thus, we were forced to reduce the size of our RBS library down to 3*2*3. Due to time constrain, we only managed to successfully introduce six combinations out of 18 into the △fimA strain, but we still obtained some optimistic results at the end. As the table and the graph shows, with the optimum mutation, RBS4, productivity was successfully increased by 34.1%. You can see the RBS information for each mutant in the below figure. In this part, we have achieved a further increase in BIOT production, thereby reducing its costs and promoting its further application in the IoT field.

Figure 2: The 3x2x3 RBS library combinations (a) and the sequence of each designed RBS (b).

Protein engineering to improve power efficiency

Second，Power Efficiency Matters!

Here, in this section, we were aiming to apply protein engineering technology based on two strategies to modify the protein monomer of e-PN, hoping to increase its voltage level.

1. Increase the number of carboxyl groups on nanowires.

One of the perspectives we considered was the number of carboxyl groups on nanowires. 

We established mathematics models to explore the relationship between the number of carboxylic groups and the output voltage. As you can see in our Model Page, when the humidity is at the same level, the potential value is proportional to the number of carboxyl groups. By increasing the number of carboxyl groups on the e-PN monomer, we can theoretically improve the potential of the basic BIOT module. 

Figure 4: The model for BIOT potential generation.

To verify our idea, we designed 16 mutants of the e-PN gene, to increase the number of carboxyl groups which reacted with water molecules to form hydrogen ions. We made it by replacing 1 to 4 amino acids in non-conservative regions with Aspartic acid or Glutamic acid. That's because these two amino acids both have two carboxyl groups. The amino acids we chose to replace had similar structures to them based on the Grantham's distance, according to our research.

Figure 5: Aspartic acid (Asp) and Glutamic acid (Glu) both have extra -COOH.

Figure 6: Increase the potential value of BIOT by adding more Carboxylic groups.

You can see the mutated site of mutants with 1-4 carboxylic groups added below.

Table 1: The mutant sites of mutants with 1-4 Carboxylic groups added.

2. Decreasing the diameter of the nanowires.

Another design we considered was to decrease the diameter of the nanowires. By decreasing the diameter, it becomes harder for the water molecules to pass through the nanopores; it will lead to greater water potential gradient as reported from the work published on Nature. 

Through literature search, we found two mutations F51W and Y57W can make the diameter of nanowires decrease from 3nm to 1.5nm.

Figure 7: Increase the potential value of BIOT by decreasing the diameter of nanowires.

These mutations mean changes to the structure. Due to the huge workload of determining the performance of each mutant, We first used Swiss Model to predict the structure of every mutant to examine whether the mutations affect the structure dramatically and then analyze the Root-mean-square deviation of atomic positions, RMSD, between wild-type and mutants using PyMOL. The smaller the value is, the more similar they are. The results were all below 0.03, far lower from the threshold, which confirmed that our design would work successfully.

Figure 8: RMSD value of the mutants compared to the wildtype nanowires.

Then we clone, transform and purify of the mutants and test their performance. From the chart of the output voltage of different mutated proteins, we can see that the 4A mutant gave a rather ideal result, with the voltage increased by 46%. It reached 0.51V compared to the original 0.35V! It's definitely an essential step in our project. The output of the thinner mutant THIN was 0.47V, which also increased from the original voltage by 34.3%. It means that the two designs both work as expected.

Figure 9: The Potential Value of different mutants.

Since there's an obvious increasing trend from 1A to 4A & THIN, we did a repetitive measurement with these five mutated proteins. Surprisingly, the difference was narrowed, but the result was enhanced generally. We admit that the output voltage of the mutated protein is still unstable, and the reason behind it needs further analysis. But anyway, it provides a new idea for future study.

This part shows the best combination effect, with the optimum RBS mutation RBS4 which increased the protein productivity by 34.1%(figure 2), and the optimum pilA mutation 5A which increased the electricity productivity by 46%(figure 9).

References

[1] Liu X, Gao H, Ward J E, et al. Power generation from ambient humidity using protein nanowires[J]. Nature, 2020, 578(7796): 550-554.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 2393
  Illegal BglII site found at 2857
  Illegal BglII site found at 4658
  Illegal BamHI site found at 1073
  Illegal BamHI site found at 3182
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1300
  Illegal AgeI site found at 1146
  Illegal AgeI site found at 1439
  Illegal AgeI site found at 2002
  Illegal AgeI site found at 4626
  Illegal AgeI site found at 6914
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 7920
  Illegal SapI.rc site found at 4262