Part:BBa_K5115066

mineral, U module

Introduction

Most of the U module is the same with BBa_K5115067（mineral, F module）, except for that in U module the EP is combined with hoxU and in F module the EP is combined with hoxF. We design these two parts to compare out which subunit for EP to combine with is more effective. The final experiment shows that module F works better than module U. For more details about the module and experiment, please check BBa_K5115067（mineral, F module）.

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 1,3-8: Amplification of specific regions corresponding to hoxF, hoxY, hoxH, hoxW, hoxI, hypA, and hypB, demonstrating successful assembly and integrity of the ribozyme-connected hox and hyp, without hoxU (no band in lane 2) as designed. 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. As a result, the Hydrogenase-carboxysome function test can only be conducted in E.coli with F module. Please visit mineral F modulefor results of this experiment.

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 OD_530nm 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 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 11. 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

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 5912
  Illegal BglII site found at 6730
  Illegal BglII site found at 7023
  Illegal BglII site found at 15401
  Illegal BglII site found at 15479
  Illegal BamHI site found at 7317
  Illegal XhoI site found at 5357
  Illegal XhoI site found at 7524
  Illegal XhoI site found at 9763
  Illegal XhoI site found at 11592
  Illegal XhoI site found at 12754
  Illegal XhoI site found at 14593
  Illegal XhoI site found at 15409
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 5791
  Illegal NgoMIV site found at 6329
  Illegal NgoMIV site found at 6539
  Illegal NgoMIV site found at 6851
  Illegal NgoMIV site found at 7617
  Illegal NgoMIV site found at 8297
  Illegal NgoMIV site found at 10904
  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 12079
  Illegal AgeI site found at 15362
  Illegal AgeI site found at 16361
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 9417
  Illegal BsaI site found at 15183
  Illegal BsaI site found at 15345
  Illegal BsaI.rc site found at 5960
  Illegal BsaI.rc site found at 6464
  Illegal SapI site found at 266
  Illegal SapI.rc site found at 15294