Part:BBa_K4225007
Pc-pelB-rDAO-6xHis
This is a composite part that consists of rDAO that is tagged with N-terminal pelB translocation tag (BBa_J32015) and induced by a constitutive promoter Pc (BBa_J23109). The presence of pelB allows the rDAO enzyme to be translocated into the periplasm of E.coli which allows better formation of disulfide bonds due to the presence of DsbA-DsbB and DsbC-DsbD systems. Note that the pelB translocation tag doesn’t influence the catalysis function of the rDAO, and thus rDAO will catalyze the conversion of diamine to H2O2 and other products. Teams that are planning to use bioamine as an input signal, such as histamine or cadaverine, may use this enzyme to convert the input to H2O2, which can activates many H2O2 inducible promoter such as oxySp(BBa_K4225001) and katGp(BBa_K4225004) with the assistance of OxyR protein(BBa_K1104200) as their transcription factor.
C-terminal 6xHis is added for characterization using western blotting.
Contribution: HKUST 2022
Characterization of pelB-rDAO with Ninhydrin and Histamine Assay
Summary
We use ninhydrin and histamine to measure the activity of rDAO to decrease the concentration of histamine. From the experiment, we would expect a decrease of ninhydrin absorbance using pelB-rDAO compared to the negative control.
Experiment
pelB-rDAO (BBa K4225007) constructs are tested with 600 ppm of histamine alongside Pc-rDAO (BBa K4225021) as a positive control and pSB1C3 as a negative control. Constructs are inoculated in 5mL LB with chloramphenicol (CHL LB) for 16 hours. The constructs are then back diluted to 1.0 OD600. These steps are done to equalize the amount of cells for each culture. They are then added to 600 ppm of histamine and incubate for 1 hour. Before and after incubation for 1 hr, the samples are spun down. Afterwards, the supernatants are taken, mixed with ninhydrin and heated at 80oC for 20 min. Finally, the samples are placed in an ice box for 3 minutes and absorbance is measured at 570 nm.
Results and Discussion
Statistically significant decrease (p<0.01, p<0.03 and p<0.01) of absorbances can be seen with pelB-rDAO from 0 to 1 hr, 1 to 2 hr and 0 to 2 hr respectively. On the other hand, no statistically significant decrease can be seen for the rDAO construct for every hour, and only a statistically decrease (p<0.03) can be seen for 0 to 2 hr result. Moreover, for our -ve construct, no significant decrease or increase can be seen. Thus, the evaporation of histamine and the effect of bacterial culture towards the result of our experiment could be ignored. Therefore, we can state that the decrease of absorbance, which is related to the concentration of diamine, in pelB-rDAO and rDAO construct is due to the rDAO enzyme. Knowing that the function of rDAO is to catalyze the conversion of diamine to H2O2, then this result indirectly proves that H2O2 is also produced due to the rDAO enzyme.
The decrease of the percentage of absorbance for pelB-rDAO construct are: -17.8% for 0 hr to 1 hr, -6.59% for 1 to 2 hr, and -23.2% for 0 hr to 2 hr, while for rDAO construct are: -6.59% for 0 hr to 1 hr, -7.19% for 1 hr to 2 hr and -10.8% for 0 hr to 2 hr. Comparing just the time point with the statistically significant result, which is 0 to 2 hr, then the pelB-rDAO construct has a larger decrease of percentage of absorbance compared to the rDAO construct ( -23.2% v/s -10.8%). This demonstrates that there is a higher activity of rDAO in the bacteria with pelB-rDAO compared to the latter, proving that the pelB tag functions like we hypothesised. For more information on the effect on pelB on rDAO in our DH5α chassis, refer to our wiki page.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1172
Illegal BamHI site found at 2100
Illegal BamHI site found at 2354
Illegal XhoI site found at 970 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 156
- 1000COMPATIBLE WITH RFC[1000]
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