Part:BBa_K5115067

mineral, F module

Introduction

This part is made up of BBa_K5115063(hox and hyp, with EP targeted hoxF) and BBa_K5115060(ribozyme+RBS+cso without csoS3)+stem-loop). The hoxs and hyps make up the Ni-Fe hydrogenase and the csos make up the carboxysome. In our design, the hydrogenase is encapsulated into the carboxysome by EP. All the subunits of the proteins constructed into our ribozyme-assisted polycistronic co-expression system. We introduced this ribozyme-assisted polycistronic co-expression system from 2022. By inserting ribozyme sequences between CDSs in a polycistron, the RNA sequences of Twister ribozyme conduct self-cleaving, and the polycistronic mRNA transcript is thus co-transcriptionally converted into individual mono-cistrons in vivo.

With this design, we achieve co-expression of hydrogenase, carboxysome and EP at similar level. These parts make up the Ni-Fe hydrogenase with the hoxF targeted by EP and carboxysome. The former will be directed into the latter, creating a stable environment for the Ni-Fe hydrogenase to work in.

The Ni-Fe hydrogenase we use is an enzyme that functions in vivo bidirectionally for NAD⁺ reduction and NADH oxidation coupled to H₂ uptake and H₂ production, respectively. In our design, the Ni-Fe hydrogenase works mainly to restore the nickel to a zero valence, which can help reduce nickel toxicity and collect nickel particles. The Ni-Fe hydrogenase is made up of six major and three auxiliary subunits. The former includes hoxF, hoxU, hoxY, hoxH, hoxW and hoxI, while the latter includes hypA, hypB and hypF.

The hoxF and the hoxU form the module of NADH dehydrogenase. The hoxF is a hydrogenase subunit responsible for electron transport. The most important group in hoxF is FMN-b, which has the ability of switching electron. Under anaerobic conditions, NADH is oxidized to NAD⁺ on the surface of hoxF subunit. In the meanwhile, the electrons generated in this reaction travel through a series of processes to the hoxH, completing the reduction of the hydrogen ion. Under aerobic conditions, NAD⁺ is reduced to NADH on the surface of the hoxF subunit. The electron transferring is contrary to former. The hoxU houses a 2Fe-2S cluster and is responsible for the role of conducting electrons between hoxH and hoxF.

The hoxY and the hoxH form the module of catalytic center.The hoxY houses a [4Fe-4S] cluster of the site, and an FMN group (FMN-a) near the Ni-Fe site in the hoxH. It is also responsible for the role of conducting electrons between hoxH and hoxF. The most important site in hoxH is the [NiFe] -hydrogenase active site, which is composed of Ni and Fe particles coordinated with cysteine residues, cyanide and carbon monoxide. It is the most central component of our intracellular conversion of nickel ions. On its surface, oxidation and reduction of hydrogen gas happens alternately according to different oxygen status.

The rest of the subunits may work together to ensure that the hydrogenase can assemble and function well. It's worth noting that hypA and hypB can cooperate to precisely guide and insert the nickel ions into the hydrogenase catalytic center.

Through the synergistic integration of the hox and hyp subunits, our system effectively enhances hydrogen production and enables the reduction of nickel ions into nanoparticles, thereby maximizing the efficiency of nickel recovery from industrial wastewater.

The EP sequence encodes an endogenous encapsulation peptide, which plays a crucial role in directing external proteins into bacterial microcompartments like carboxysomes. EP supports the functional assembly of the carboxysome, enhancing the stability and activity of encapsulated enzymes. This targeting mechanism is essential for protein encapsulation within these structures, aiding in the assembly of a functional, proteinaceous shell that sequesters enzymes or other proteins, ensuring efficient catalysis or protection from environmental stress.

The csoS operon, originating from the Halothiobacillus neapolitanus, encodes a series of proteins essential for the assembly of α-carboxysomes, a type of microcompartment that facilitates the sequestration and concentration of enzymes involved in carbon fixation, particularly ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). In literature, α-carboxysomes have been extensively studied and successfully utilized in Escherichia coli for enhancing carbon fixation efficiency and optimizing metabolic pathways. The csoS operon includes key structural proteins including csoS4B, csoS1C, csoS1A, csoS1B, csoS1D, csoS4A, and CsoS2, which play crucial roles in forming the shell and encapsulating cargo enzymes, including those required for hydrogen production. The operon serves as a model for synthetic biology applications, particularly in constructing nanoreactors capable of enhancing catalytic functions through encapsulation of heterologous enzymes. The successful expression of this operon in E. coli demonstrates its potential for industrial and biotechnological applications, enabling the creation of efficient microbial systems for sustainable bioprocessing.

Usage and Biology

The F module harnesses the collaborative power of hydrogenase enzymes, carboxysome compartments, and encapsulation peptides to drive an innovative approach for nickel reduction in E. coli. This integrated module not only advances the biotechnological potential of engineered microorganisms but also addresses environmental concerns related to nickel contamination by converting harmful ions into less toxic nanoparticles.

Characterization

Agarose gel electrophoresis

We firstly make sure that our genes are successfully introduced into the E.coli

In our design, we choose to remove the subunit csoS3 from the cso operon. Previous studies have shown that it is not essential for carboxysome assembly or function. Deleting this subunit can release some burden of the engineered E.coli. The result of PCR shows that the removal of csoS3 was rather successful.

Figure 1. Agarose gel electrophoresis of PCR products amplified from E. coli (DH5α) colonies. M: DNA Marker. (A) Lanes 1-8: Amplification of specific regions corresponding to csoS2, csoS3, csoS4A, csoS4B, csoS1C, csoS1A, csoS1B, and csoS1D, demonstrating the presence of the expected subunits derived from the α-carboxysome plasmid. (B) Lanes 1-8: Primers as in (A) were used for amplification. Please note no specific band in lane 2, which is due to the removal of csoS3 from the operon. Also, bands in (B) 3-8 are all smaller than (A) 3-8. Primers for these PCR are listed on https://2024.igem.wiki/fudan/parts.

To have our Ni-Fe hydrogenase directed into the carboxysomes, we deleted hoxF from the original hydrogenase operon and create another part to accommodate the hoxF-GS-EP which wrapped by ribozyme-assisted polycistronic co-expression system (pRAP) sequences. We run this PCR test to examine the result of the hoxF removal.

Figure 2. Agarose gel electrophoresis of PCR products amplified from one E. coli (DH5α) colony. M: DNA Marker. Lanes 1-8: Corresponding bands for hoxF, hoxU, hoxI, hoxH, hoxW, hoxY, hypA, and hypB, demonstrating successful assembly and integrity of the ribozyme-connected hox and hyp operon as designed. Primers for these PCR are listed on https://2024.igem.wiki/fudan/parts.

Figure 3. Agarose gel electrophoresis of PCR products amplified from one E. coli (DH5α) colony. M: DNA Marker. Lanes 2-8: Amplification of specific regions corresponding to hoxU, hoxY, hoxH, hoxW, hoxI, hypA, and hypB, demonstrating successful assembly and integrity of the ribozyme-connected hox and hyp, without hoxF (no band in lane 1) as designed. Lane 4, the band for hoxH appears faint, pointed by the red arrowhead. Lane 7, there are non-specific bands, besides the red arrowhead pointed hypA. Primers for these PCR are listed on https://2024.igem.wiki/fudan/parts.

Fluorescence microscopy results

Our experiments validated the role of EP by first fusing it with the StayGold fluorescent protein to assess its effectiveness in directing protein localization to the α-carboxysome. The fluorescence microscopy results confirmed successful incorporation of StayGold into the carboxysome shell, indicating that EP effectively directs proteins into the compartment. Subsequently, we incorporated EP with core assembly proteins of hydrogenase to examine nickel particle formation. Analysis of nickel particle distribution within E. coli revealed that EP facilitated the assembly of the α-carboxysome shell around the hydrogenase, leading to the formation of well-defined nickel particles. These results demonstrate that EP not only directs protein localization but also supports the functional assembly of the carboxysome, enhancing the stability and activity of encapsulated enzymes.

Figure 4. Fluorescence images of E. coli expressing stayGold fused with EP, without or with cso-S3. Images were captured using spinning disk confocal with a 150x objective lens, as described on our Experiments page. Bacteria in A-C only express stayGold fused with EP BBa_K5115057, while bacteria in D simultaneously express BBa_K5115057 and BBa_K5115065. 1 mM IPTG was added to A,B only. Images without scale bar are 5x5 µm square, unless specifically indicated below. (A) The entire image field is shown (41.27x41.27 µm square), with brightfield image on the left, and green fluorescence image on the right. (B) Four regions in (A) are enlarged, showing uniform distribution of green fluorescence. (C) Although no IPTG was added, leaky expression from the promoter is sufficient to fill bacteria with green. (D) With all carboxysome components expressed, stayGold fused with EP concentrated to the carboxysome. Leaky expression from the promoter is sufficient to drive 1 or 2 carboxysome formed within each bacteria.

Hydrogenase-carboxysome function test

While culturing the bacteria, four biological replicates were performed for each condition. If induced with 1 mM IPTG, None of the four bacteria with U module was able to grow during overnight culture, only 1 F module grow. We chose this growing F-module-bacteria to do further experiment.

We tried to run a protein electrophoresis to show the protein translation status. However, we can't find distinct bandings on the protein gel. We suspect that this failure results from too much burden the plasmid has brought to our E.coli. To learn more about the burden, please visit our software wiki. Convinced by our approaches, we sent our sample to the EM Core Facility for electron microscopy examination. The EM result is shown.

Figure 5. Transmission electron microscopy images of negative stained E. coli expressing MINERAL F module. Osmium tetroxide and uranyl acetate were used for the staining A-E. Scale bar shown on the image. (A) Overview of E. coli cells. (B) Sections of bacteria, filled with carboxysome-sized regions (CSR) surrounded by electron dense dots. In one cell, all visual CSR are circled by yellow dash lines. (C) The size of CSR are various, with two examples circled. (D,E) For cells with less electron dense dots, CSR are clear, with the cell in C fully packed with CSR and the cell in D sparsely packed. (F) No uranyl acetate staining. The image confirms that the electron dense dots throughout A-E are not salt crystals but actual metallic particles, which we believe are Ni particles. Three 80-nm square regions are enlarged, showing polyhedral outline of CSR, with Ni particles surrounded.

Ni uptake upon expressing module F/U

We wanted to compare the nickel ion absorption capacity of bacteria introducing F or U modules under different induction conditions.

We first fill the flask with enough hydrogen to ensure that the hydrogenase can reduce nickel ions rather than oxidize them.

Figure 6. Inject hydrogen into the culturing flask

Before we started the Ni uptake experiments, we set two no-induction groups of nickel-containing solution to culture bacteria with F or U module, two bottles in each group. It turned out that there was an uninduced bacteria with F module whose medium color was inconsistent with the others. After excluding other possible causes, we speculate that instead of showing the magnitude of the nickel ions' concentration, the bluish color in the cultural media might due to a specific absorption when bacteria have certain number of nickel particles, which we called leaky expression.

Figure 7. The four flasks culturing F-module or U-module bacteria

To calculate the absorption of nickel, we eventually chose to measure the ODs using spectrophotometric methods.

Figure 8. Comparison of Ni²⁺ Uptake Efficiency by Different E. coli The graph shows the percentage of Ni²⁺ concentration absorbed by E. coli expressing indicated modules (E. coli strain: BL21 DE3). Ni²⁺ uptake was calculated based on the difference between initial and final concentrations in the supernatant, divided by 100 mg/L. The single bacteria colony was picked and grown overnight to reach optical density (OD₆₀₀) > 1. Prepare a sealed 25-mL LB culture in a 250-mL bottle, with: 100 µL overnight bacteria liquid culture, 25 µg/mL Kan, 1 mM methyl viologen dichloride, 100 mg/L NiCl2, bubbled with ~250 mL 5.6% hydrogen gas (slowly, with hand-shaking, about 5 minutes). Culture for 30 hours, at 37°C with a rotating speed at 220 rpm. Four biological replicates were performed for each condition, and error bars represent the standard errors of the means (SEM) of these replicates. Plain BL21 DE3 was used as control. None of the four bacteria with U module was able to grow during overnight culture if induced with 1 mM IPTG, only 1 F module grow. Additional 1 mM IPTG was added into the 25-mL culture of "F induced". ANOVA test shows that all constructs increase Ni²⁺ uptake significantly compared to the control (U module, p = 0.0045; F module, p < 0.0001). P value was calculated using Dunnett’s post-test.

Nematode experiment

In this year, we have innovatively employed Caenorhabditis elegans as environmental indicator organisms to assess the potential environmental impact of our project. To learn more about Caenorhabditis elegans, please visit our safety wiki.

By feeding nematodes with the standard E. coli strain OP50 and engineered bacteria containing nickel particle products and comparing their locomotion behavior, we found no significant difference in movement between the control group fed OP50 and the experimental group. This indicates that our product is harmless to nematodes, making it environmentally and biologically friendly.

Figure 9. Representative swimming images of Caenorhabditis elegans fed with E.coli strain OP50. The E.coli strain: BL21 DE3, induced with 1 mM IPTG, cultured at 37°C for 30 hours in 100 mg/L Ni2+ solution under 5% hydrogen catalysis. The images are displayed at six times the normal speed.

Figure 10. Representative swimming images of E.coli introduced with the hydrogenase core component HoxF in a carboxysome. The E.coli strain: BL21 DE3, induced with 1 mM IPTG, cultured at 37°C for 30 hours in 100 mg/L Ni2+ solution under 5% hydrogen catalysis. The images are displayed at six times the normal speed.

Figure 11. Representative swimming images of E.coli introduced with the hydrogenase core component HoxU in a carboxysome. The E.coli strain: BL21 DE3, induced with 1 mM IPTG, cultured at 37°C for 30 hours in 100 mg/L Ni2+ solution under 5% hydrogen catalysis. The images are displayed at six times the normal speed.

Figure 12. Comparison of the average distance moved per minute and the average turning angle per turn of Caenorhabditis elegans. The nematodes were fed with E. coli strain OP50, E. coli containing the hydrogenase core component HoxF in the carboxysome, and E. coli containing the hydrogenase core component HoxU in the carboxyl matrix (strain: BL21 DE3, induced with 1 mM IPTG, cultured in a 100 mg/L Ni2+ solution under 5% hydrogen catalysis at 37°C for 30 hours). Five L2-stage nematodes were picked for each plate and cultured at 20°C for 18 hours. For each dataset, at least three independent nematode images were collected, and more than 1 minute of movement was recorded using a Nikon D850 camera at 1080p 30fps, yielding at least 1900 data points, analyzed by ImageJ Plugins Animal Tracker.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 208
  Illegal NotI site found at 5126
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 366
  Illegal BglII site found at 5743
  Illegal BglII site found at 5821
  Illegal BglII site found at 14467
  Illegal BglII site found at 15285
  Illegal BglII site found at 15578
  Illegal BamHI site found at 6214
  Illegal XhoI site found at 5751
  Illegal XhoI site found at 5943
  Illegal XhoI site found at 6421
  Illegal XhoI site found at 8660
  Illegal XhoI site found at 10489
  Illegal XhoI site found at 11651
  Illegal XhoI site found at 13490
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 6514
  Illegal NgoMIV site found at 7194
  Illegal NgoMIV site found at 9801
  Illegal NgoMIV site found at 9908
  Illegal NgoMIV site found at 10178
  Illegal NgoMIV site found at 11024
  Illegal NgoMIV site found at 11256
  Illegal AgeI site found at 874
  Illegal AgeI site found at 1825
  Illegal AgeI site found at 2506
  Illegal AgeI site found at 3460
  Illegal AgeI site found at 5704
  Illegal AgeI site found at 10976
  Illegal AgeI site found at 16361
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 5525
  Illegal BsaI site found at 5687
  Illegal BsaI site found at 8314
  Illegal BsaI.rc site found at 14515
  Illegal BsaI.rc site found at 15019
  Illegal SapI site found at 266
  Illegal SapI.rc site found at 5636