Part:BBa_K3796207
Patp2-rbs-gfp-terminator
This part is composed of an inducible promoter Patp2, a rbs sequence in Corynebacterium glutamicum (BBa_K2963006), the fluorescent protein gene bjGFP, and the rrnB T1 and T2 terminators (BBa_K3796213). P-atp2 is an alkali inducible promoter found in Corynebacterium glutamicum. Under high pH, the gfp will have higher expression, leading to stronger fluorescence intensity.
Characterization
Patp2 is an alkali-induced promoterin our composite kill switch. With this part, we aim to verify that this promoter can respond to pH stress in Corynebacterium glutamicum and we also want to document its response towards different alkalinity.</p>
We want to test the strength of the promoter by reporter genes as well. Here we choose to use GFP because it is more stable to pH changes. The genetic circuit we used as a testing device is BBa_K3796207. Particularly, we destroyed the tac promoter on the plasmid pXMJ19 to get rid of its influence this time. Ideally, the fluorescence intensity/OD600 should be higher in relatively high pH.
We build the gene circuit by ClonExpress II one-step cloning kit (Vazyme Biotech, China). Particularly, we destroyed the tac promoter on the plasmid pXMJ19 to get rid of its influence.We used ClonExpress II one-step cloning kit (Vazyme Biotech, China) to build the circuit for testing. The expression vector was transformed into E. coli DH5α first and then into C. glutamicum by electroporation. Electrophoresis showed that we had built the circuit successfully.
After sequencing and amplifying, that vector as well as unmodified pXMJ19 were transferred into Corynebacterium glutamicum separately and cultivated on plates. We continued to use the fluorescence intensity/OD600 as a measurement data. After cultivating the bacteria in the LB liquid medium at 100 rpm, 30℃ for 12h, the OD600 of the two bacteria were adjusted to nearly the same (nearly 2.0) and inoculated in the pH gradient LB liquid medium. After a cultivation of 24h, the bacteria solution was collected and washed with PBS. Then we measured its fluorescence intensity by HITACHI F-7000 according to the excitation light of 488nm and emission light of 507nm and OD600.
Through two-way ANOVA, we can know that there’s significant difference between the control group and experiment group under different pH conditions. The relative fluorescence intensity of the control group is relatively stable at the pH range of 7-9.5, while there is a significant increase of relative fluorescence intensity in experiment group. Data shows that the difference of relative fluorescence intensity between the two groups remains high between pH=8.5 and pH=9.0, and reaches its peak near pH=9.0, indicating that the promoter Patp2 has the most ability to enhance its downstream gene expression in this pH range. Also, we can see a sharp drop of the relative fluorescence intensity in the experiment group when the pH is more than 9.5. We assume that this is because ,C. glutamicum can’t tolerate such a high alkalinity stress and the OD600 decreases a lot, making the relative fluorescence intensity seems abnormal or irregular. Three parallel experiments were done later, which supported our opinions.
We finally come to the conclusion that Patp2 can respond to pH changes in C. glutamicum, and high alkalinity can improve the expression of the downstream genes of Patp2. According to our data, the peak occurs near pH=9.0, which happens to fall in the range of the alkalinity of saline-alkaline soil. The strength pf Patp2 under higher pH needs to be further tested. Based on those results, this part can be used as a reporter to alkalinity.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 14
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 14
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 14
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 14
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 14
- 1000COMPATIBLE WITH RFC[1000]
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