Part:BBa_K2890006

HxlR + eRFP + FDM

A formaldehyde detection and degradation machine, using the combination of HxlR, eRFP and FDM.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1479
  Illegal NheI site found at 1502
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 2454
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1176
  Illegal AgeI site found at 1288
  Illegal AgeI site found at 1741
  Illegal AgeI site found at 2053
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1585
  

Usage and Biology

The composition of subpart of BBa_K2890006 is shown in Figure 1.

Figure 1 Composition of part BBa_K2890006.

Results and Discussion

As shown in Figure 2a, we can clearly see in the growth curve that as the concentration of formaldehyde increases, the growth curve of the control group decreases significantly, and the growth of the transferred eRFP bacteria is inhibited when the formaldehyde concentration rises to 6.4 mM. On the other hand, the bacteria almost stopped growing. While, comparatively, the growth curve of TEST group is basically normal and, as the concentration of formaldehyde increases, gradually slows down.

Compared the TEST group with CONTROL group, it is obviously revealed that the tolerance of bacteria to formaldehyde is spectacularly strengthened after adding this part (BBa_K2890006), which proves the degrading potential to formaldehyde.

Figure 2 Growth curve of Escherichia coli under different formaldehyde concentrations: (a) control group, (b) test group.

We can quantify the production of eRFP by means of measuring the fluorescence intensity under excitation wavelength of 575 nm. As shown in Figure 3a, with the extension of culture time, the fluorescence intensity of eRFP gradually increased which indicates that the expression level of eRFP in the medium gradually increased. In other words, the formaldehyde detection function of this working system was working normally.

Besides, as shown in Figure 3b, when the formaldehyde concentration is between 0-1.6 mM, the fluorescence intensity was gradually increased with the formaldehyde concentration increasing after culturing for 8 hours. However, after culturing for same times, the fluorescence intensity of eRFP is gradually decreased when the concentration exceeds 1.6 mM. It is proved that the formaldehyde degradation function of this working system was working as we expected.

Figure 3 Curve of fluorescence intensity and culture time (a) and photos of E. Coli after culturing for 8 hours (b) under different formaldehyde concentrations.

Followed experience is supplemented by 2019 iGEM Worldshaper-Shanghai.

Experimental design and Discussion

For characterization part, we have re-characterized the biobrick of BBa_K2890006, a device used to detect and degrade formaldehyde. We transformed the part BBa_K2890006 and remeasured its performance of detecting formaldehyde followed by iGEM’s standard measurement protocols. We hope that through our effort this biobrick can be more measurable and reproducible.

Firstly, we established a particle standard curve and fluorescein standard curve to standardize the experiment (Figure 1). By this way, we hope to eliminate the deviations caused by different laboratories and individuals when the functions of engineered strains are measured.

Figure 1 Particle standard curve (left) and Fluorescein standard curve(right).

In cell measurement part, LB is used as the blank control and empty plasmid as the negative control. Combined with test device, their performance of detecting formaldehyde have been done. In the previous experience, we found that the test group containing the working plasmid (BBa_K2890006) could efficiently survive in the culture media containing formaldehyde. Using the associated Excel data analysis template provided by iGEM measurement committee, the eRFP fluorescence value is standardized as the NET Fluorescein a.u.. Similarly, OD600 is standardized as the Net Abs600. Detailed results are as follows.

As shown in Figure 2, we can see that with the increase of the concentration of formaldehyde, the growth curve of the control group (Figure 2(B)) decreased significantly; while the growth curve of test group (Figure 2(A)) was basically normal. Compared with control group, the bacteria of the test group exhibited higher tolerance to formaldehyde especially at the concentration above 1.6 mM. It could be inferred that the test group had the good potential in degrading formaldehyde.

Figure 2. The growth curve of test group (A) and control group (B)

We also tested the production of eRFP by measuring the fluorescence intensity under the excitation wavelength of 575 nm. The results matched the previous study. According to Figure 3, the fluorescence intensity increased with the increase of formaldehyde concentration, indicating that the module of formaldehyde detection functioned well.

Figure 3. Curve of fluorescence intensity and culture time of the test group

Conclusion

In order to eliminate the deviations caused by different laboratories and individuals when the functions of engineered strains are measured, we recharacterized the working plasmid BBa_K2890006 followed by iGEM’s standard measurement protocols. According to our study, it is proven that the biobrick BBa_K2890006 is available and reproducible, and functioning as a machine which could detect and degrade formaldehyde. Moreover, we hope that the supplementing experimental data from us can be a meaningful reference for others.