Team:XHD-Wuhan-China/Contribution
Contribution
In order to provide useful support and contribution to future iGEM, we have carried out the following two aspects of work:
1. Characterization of a related previous part BBa_K1231000 supplemented with new data.
2. Provide new data from literature for the previous part BBa_K644001.
Characterization of a related previous part BBa_K1231000
1. Aim of experiment
Based on K1231000 part, a genetic circuit Pasr-B0034-amilCP was constructed to characterize the function of this Pasr promoter.
2. Methods
2.1 Construction of Pasr-pSB1C3
We construct the following gene circuit based on the principles of synthetic biology, as shown in Figure 1.
Figure 1. constitution of Pasr-amilCP gene circuit.
We cloned the Pasr promoter from the Pasr-pUC19 plasmid, and used homologous recombination to obtain the recombinant plasmid Pasr-pSB1C3, and verified the length of the recombinant plasmid to ensure the success of the recombinant plasmid through PCR and enzyme digestion.
2.2 AmilCP expression under the control of K1231000 induced by different pH
The plasmid Pasr-pSB1C3 containing the K1231000 promoter was transformed into E. coli DH5α strain. We Cultured the DH5α bacteria containing the recombinant plasmid Pasr-pSB1C3 to the logarithmic pHase in LB medium, then took 1000ul centrifugation, discarded the supernatant, and resuspend the bacterial solution in 200ul M9 medium. M9 medium was adjusted with HCL for pH, Respectively, pH=4.5, 5, 7,and each group of pH gradient has 3 biological replicates. Regarding the spotting process, add 200ul to each hole, and the pH is 4.5, 5, and 7, respectively. The first three wells are samples, and the last three wells are M9 medium corresponding to the pH (as negative controls). The concentration of amilCP was measured by microplate reader. The measurement interval is the first hour, once every 3 minutes; the second hour, once every 10 minutes; the third hour, once every 20 minutes; the fourth hour, the fifth hour, once every 30 minutes. The concentration of amilCP (OD580) and OD600 value are measured simultaneously.
3. Results
We used the plasmid Pasr-pSB1C3 as a template, and then used PCR to obtain the corresponding Pasr+amilCP fragment (860bp), as shown in Figure 2. The length of the PCR fragment is consistent with expectations. Subsequently, the Pasr-pSB1C3 recombinant plasmid was digested with xbal, as shown in Figure 3, and the result of digestion was also consistent with expectations. All this proves the success of the recombinant plasmid.
Figure 2. The electropHerogram of the Pasr+amilCP fragment after PCR.
Figure 3. ElectropHoresis of Pasr-pSB1C3 plasmid after digestion with xbal.
The successfully recombined plasmid Pasr-pSB1C3 was transformed into E. coli DH5α strain, cultured in LB medium to logarithmic pHase, then transferred to M9 medium corresponding to pH, and continuously cultured in microplate reader for 5 hours. As shown in Figure 4, the final pH=4.5, pH=5 group of bacteria liquid showed obvious blue, pH=7 group did not show blue, indicating that Pasr has a stronger promoting ability under acid induction.
Figure 4. Characterization of the color of bacterial liquid in different pH gradients.
The normalized concentration of amilCP is OD580 divided by OD600. As shown in Figure 5, the concentration of amilCP is the highest in the condition of pH=4.5, followed by the condition of pH=5, and lowest in the condition of neutral (pH=7).
Figure 5. In different pH gradients (4.5, 5, 7), the corresponding amilCP concentration of culture for 1 hour.
The concentration of amilCP was measured several times during the continuous culture for 5 hours. As shown in Figure 6, in the first 5 minutes, E. coli was in the adaptation stage from LB to M9 medium, so the concentrationt of amilCP decreased. In 5-10 minutes, E. coli has relatively adapted to the M9 medium, its life activities have become active, and the concentrationt of amilCP has been relatively increased. The follow-up results showed that only under acidic conditions (pH=4.5, pH=5), the concentrationt of amilCP gradually increased, and under the condition of pH=4.5, the concentrationt of amilCP increased the fastest, amilCP's concentration reaches the maximum under the condition of pH=4.5. Under neutral (pH=7) conditions, the concentration of amilCP is gradually decreasing. This proves that Pasr is an acidic promoter, which has a significant activation effect under low pH conditions. After the time reaches 1 hour, the concentration of amilCP gradually decreases, which may be due to the exhaustion of nutrients in the medium, and the life activities of bacteria gradually weaken.
Figure 6. The change trend of amilCP concentration within 5 hours under different pH gradients (4.5, 5, 7).
4. Conclusion
The plasmid Pasr-pSB1C3 based on the K1231000 is sensitive to pH. Under the condition of pH=4.5, the expression of amilCP is the highest. The results indicate that the Pasr promoter is activated by low pH. We use amilCP to characterize the capability of Pasr promoters, and we can observe color changes with the naked eyes. The Pasr-amilCP system can be used as a biosensor for detecting environmental pH. The detection method is convenient and the result is intuitive.
5. References
Liene E S, Lis K S, Garbenciute V, et al. The Acid-Inducible asr Gene in Escherichia coli: Transcriptional Control by the pHoBR Operon[J]. Journal of Bacteriology, 1999, 181(7): 2084-2093.
Ogasawara H, Hasegawa A, Kanda E, et al. Genomic SELEX Search for Target Promoters under the Control of the PHoQP-RstBA Signal Relay Cascade[J]. Journal of Bacteriology, 2007, 189(13): 4791-4799.
Contribution to the previous part BBa_K644001 based on literature
Introduction
The author's research shows that mucoadhesive nanogel containing farnesol has obvious antifungal activity for Candida albicans. Then authors investigated the mechanism by which farnesol reduces the infection rate of bacteria, which involves the expression of HWP1 gene.
Results
The effect of farnesol, CS and AL nanogels containing farnesol
on the expression of HWP1, SAP6 and Rim101 genes was
investigated using real-time PCR (Figure 7). The finding shows
that expression of HWP1 and SAP6 genes in C. albicans treated
with 300 mM concentration of farnesol, CS and AL nanogels
containing farnesol decreased significantly in comparison with
un-treated control group (p<.01). However, it was found the
significant difference between the expression of SAP6 gene of
C. albicans treated with CS nanogel and non-treated Candida
was observed. The expression of Rim101 in Candida treated
with CS nanogel significantly decreased as compared to the
non-treated cells (p<.01), whereas, Rim101 C. albicans treated
with farnesol and AL nanogel containing farnesol did not
show any change in the gene expression level.
References
Fatemeh Nikoomanesh, et al.Design and synthesis of mucoadhesive nanogel containing farnesol: investigation of the effect on HWP1, SAP6 and Rim101 genes expression of Candida albicans in vitro. ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 2019, VOL. 47, NO. 1, 64–72
