Difference between revisions of "Part:BBa K2471001"
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===Improvement from CUG-China 2022=== | ===Improvement from CUG-China 2022=== | ||
| − | + | <p align="justify">This year CUG-China improved this part by switching the promotor from consititutive promotor T7 to IPTG inducible promotor Ptac, resulted in a new composited part <bbpart>BBa_K4242017</bbpart>. In this way we can not only control the <i>yhjH</i> expression level by adding IPTG, but also make it functional in a wider variety of strains who lack the T7 expression systems.</p> | |
| − | This year CUG-China improved this part by switching the promotor from consititutive promotor T7 to IPTG inducible promotor Ptac, resulted in a new composited part <bbpart>BBa_K4242017</bbpart>. In this way we can not only control the yhjH expression level by adding IPTG, but also make it functional in a wider variety of strains who lack the T7 expression systems. | + | [[Image:pSB3C5-A3.png|centre|700px]] |
| + | <b>fig.1 a</b>|Structure of control biobrick pSB3C5-Ptac <b>b</b>|Structure of pSB3C5-T7-YhjH <bbpart>BBa_K2471001</bbpart> | ||
| + | <b>c</b>|Structure of improved part pSB3C5-YhjH <bbpart>BBa_K4242017</bbpart> | ||
| + | <p align="justify">In our project, we need to overexpress the YhjH, a c-di-GMP hydrolase, which can result in the decrease of biofilm. However, this part is only functional in the chassis containing T7 RNA polymerase. Our strain, Shewanella oneidensis MR-1 doesn’t fit this condition. The biofilm formation experiment also proved that the biofilm formation of the strain containing original part didn’t reduce compared to the control group. In the group of improved part, biofilm formation was significantly reduced with the addition of inducer IPTG compared with the control group, indicating this new part is well functional in <i>S. oneidensis</i> MR-1 strain.</p> | ||
| + | [[Image:PSB3C5-Biofilm.png|centre|700px]] | ||
| + | <b>Fig. 2</b> Biofilm formation of different engineered strains of <i>S. oneidensis</i> MR-1 | ||
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Latest revision as of 12:22, 12 October 2022
(T7 promoter + RBS) + (yhjH) + (T1 terminator)
This BioBrick™ contains the necessary genetic circuitry to constitutively express the yhjH gene, that when expressed, results in the production of the yhjH enzyme. This will catalyze the reaction of c-di-GMP to GMP, which will inhibit biofilm formation and promote motility.
A chassis with the T7 polymerase gene is needed for the successful expression of this BioBrick™.
Usage and Biology
This BioBrick™ contains the necessary genetic circuitry to constitutively express the yhjH gene, that when expressed, results in the production of the yhjH enzyme. This will catalyze the reaction of c-di-GMP to GMP, which will inhibit biofilm formation and promote motility.
Characterization of yhjH gene by Tec Chihuahua
The existing biopart https://parts.igem.org/Part:BBa_K861090 (BBa_K861090), which is in iGEM's Parts Registry since 2012, was improved by adding a T7 promoter, RBS and T7 terminator to its sequence; which resulted in the creation of our new composite BioBrick® (BBa_K2471001). The function of the enzyme encoded by the yhjH gene is to promote motility whilst reducing biofilm production; to demonstrate this, Escherichia coli BL21(DE3) was transformed with the new BioBrick®, to subsequently, study bacterial adherence by performing a colorimetric absorbance study. Greater adhesion was observed per area in the transformed bacteria with the composite yhjH BioBrick® than in the normal, non-transformed ones. This is because, by promoting motility, the bacteria were able to reach more area within the wells, and thus, form more biofilm although its normal production was being suppressed. Based on this, we can affirm that the improved yhjH BioBrick® works and that it's expressed correctly in Escherichia coli BL21(DE3).
Bacteria often thrive in surface-associated multicellular communities that have advantages over individual cells, such as protection against unfavorable environmental conditions (predation, the presence of antimicrobials, host's immune response, etcetera). Biofilms are sessile communities, with microorganisms embedded within a matrix and attached to a surface (Verstraeten et al., 2008). Another mechanism for bacteria to cope with changes in the surrounding is to actively leave unfavorable environments for more fitting ones. Thus, being motile provides a survival advantage to many bacteria. The flagella present in many species provides them with an efficient form of locomotion through liquid and viscous environments, as well as on surfaces (Paulick et al., 2009).
The improvement of the yhjH gene found in iGEM's Parts Registry (BBa_K861090) was done by adding a T7 promoter and RBS https://parts.igem.org/Part:BBa_K525998 (BBa_K525998) and a T1 terminator https://parts.igem.org/Part:BBa_B0010 (BBa_B0010) to its sequence, in order to make the expression of the protein of interest possible; the result was the creation of one of our three composite BioBricks (BBa_K2471001). Escherichia coli BL21(DE3) was transformed with it in order to characterize it using an absorbance-based colorimetric protocol (to see specifications visit protocols section).
Figure 1. ELISA microplate where the colorimetric protocol was carried out.
Since the equipment was unable to correctly register the readings, a 1:10 and 1:20 dilution of the original ELISA microplate was made for the sake of recording the absorbance values. In addition, this dilution made possible to visualize the difference in colorant at sight. Only the 1:10 was taken into consideration. As shown in Figure 1, the colorant intensity is higher in the yhjH transformation than in the normal bacteria.
Figure 2. Calibration curve used to interpret the obtained absorbance values.
The calibration curve shown in Figure 2 was used to interpret the results. Using the equation generated from the curve, the OD was calculated and based on the OD obtained, both types of bacteria were classified as non-adherent, weakly, moderately and strongly adherent. The untransformed bacteria were classified as weakly adherent, while the one transformed with the yhjH BioBrick was cataloged as moderately adherent.
The function of this gene is to enhance motility whilst reducing biofilm formation. Greater adherence was observed in the bacteria transformed with our BioBrick than compared to the normal, untransformed one. This was thanks to it being more motile, which allows the bacteria to reach more area within the well. Although the biofilm formation was inhibited, the increased motility allowed the bacteria to cover a larger area, giving higher absorbance values. This is why the project makes use of two more genes, aiiA and epsE, which tackle biofilm formation and the increased motility, respectively; it's the use of the three genes that really make the whole project work correctly. Summarizing, we can state that the improved yhjH BioBrick does work and that it's correctly being expressed in the E. coli BL21(DE3).
Improvement: Macquarie University 2019
We utilised the yhjH and characterisation described by Tec Chihuahua to design our improved part, [BBa_K3151008] https://parts.igem.org/Part:BBa_K3151008. By adding inducible Lac promoter (BBa_R0010) and double terminator (BBa_B0015), our construct is not only able to maintain inducibile exression with IPTG, but can be transformed into a wider variety of E. coli strains, including those which lack the T7 RNA polymerase, such as DH5α and Nissle 1917.
We characterised and compared the original and improved biopart by assaying biofilm formation through spotting cells onto LB plates supplemented with Congo Red (to stain cellulose) and Coomassie Blue (to stain curli fimbriae)[1]. Nissle 1917 cells transformed with the improved biopart (Fig. 1C) show significant reduction in cellulose biosynthesis compared with the negative control Nissle 1917 cells (Fig. 1A), indicating significant phosphodiesterase activity. Nissle 1917 cells transformed with the original biopart (Fig. 1B) with show no significant reduction in cellulose biosynthesis compared with the negative control Nissle 1917 cells (Fig. 1A).
Figure 1: Spot Assay of the negative control Nissle 1917 (A), Nissle 1917 with T7+yhjH (B), and Nissle 1917 with Lac+yhjH (induced with 1mM IPTG) (C). Plate observed under blue light to enhance visualisation of Congo Red-stained cellulose.
- Cimdins A, Simm R, Li F, Lüthje P, Thorell K, Sjöling Å, Brauner A, Römling U. Alterations of c‐di‐GMP turnover proteins modulate semi‐constitutive rdar biofilm formation in commensal and uropathogenic Escherichia coli. MicrobiologyOpen. 2017 Oct;6(5):e00508.
References
Paulick A., et al. (2009) Two different stator systems drive a single polar flagellum in Shewanella oneidensis MR-1. Mol. Microbiol. 71:836–850 [1]
Verstraeten N., et al. (2008) Living on a surface: swarming and biofilm formation. Trends Microbiol. 16:496–506 [2]
Improvement from CUG-China 2022
This year CUG-China improved this part by switching the promotor from consititutive promotor T7 to IPTG inducible promotor Ptac, resulted in a new composited part BBa_K4242017. In this way we can not only control the yhjH expression level by adding IPTG, but also make it functional in a wider variety of strains who lack the T7 expression systems.
fig.1 a|Structure of control biobrick pSB3C5-Ptac b|Structure of pSB3C5-T7-YhjH BBa_K2471001 c|Structure of improved part pSB3C5-YhjH BBa_K4242017
In our project, we need to overexpress the YhjH, a c-di-GMP hydrolase, which can result in the decrease of biofilm. However, this part is only functional in the chassis containing T7 RNA polymerase. Our strain, Shewanella oneidensis MR-1 doesn’t fit this condition. The biofilm formation experiment also proved that the biofilm formation of the strain containing original part didn’t reduce compared to the control group. In the group of improved part, biofilm formation was significantly reduced with the addition of inducer IPTG compared with the control group, indicating this new part is well functional in S. oneidensis MR-1 strain.
Fig. 2 Biofilm formation of different engineered strains of S. oneidensis MR-1
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]