Part:BBa_K4799989
pAtPCR2
pAtPCR2 is the promoter we extracted from the AtPCR2 gene.
Usage and Biology
The AtPCR2 gene, or "Arabidopsis thaliana plant cadmium resistance 2" gene, is found in the model plant species Arabidopsis thaliana. This gene is capable of providing resistance to cadmium, a toxic heavy metal, which normally would accumulate in leaves and cause phytotoxic effect on plants, such as change in chloroplast structure and mass production of reactive oxygen species, followed by oxidative stress and eventual cell death.
The AtPCR2 gene belongs to the PLAC8 motif-containing/PCR gene family. These genes all encode for proteins that facilitates various biological processes, including stress responses and heavy metal detoxification. As mentioned above, the AtPCR2 gene plays a crucial role in protecting plants against the harmful effects of cadmium toxicity. When plants are exposed to cadmium, the expression of the AtPCR2 gene is up-regulated, leading to the production of the AtPCR2 protein. This protein is believed to be involved in transporting or detoxifying cadmium ions within plant cells. Thus, accumulation of cadmium and its toxic effects on plant development are prevented.
Design considerations
When the carrier of this promoter is exposed to cadmium, pAtPCR2 promoter is supposed to initalize following coding regions that are connected to this promoter, thus expressing the gene.
Reference: Reddy, C.S., Cho, M., Kaul, T., Joeng, J.T., & Kim, K.M. (2023). Pseudomonas fluorescens imparts cadmium stress tolerance in Arabidopsis thaliana via induction of AtPCR2 gene expression. Journal of Genetic Engineering & Biotechnology, 21. doi: 10.1186/s43141-022-00457-7.Build
Please refer to the part page for BBa_K4799990 (pAtMRP3) from our team this year(2023) for detailed promoter amplification, E.coli transformation, Agrobacterium transformation, and functional verification design. Particularly, based on the results of pAtMRP3, we add a GFP report experiment for AtPCR2 promoters to get more complete evidence on whether this promoter could respond to cadmium treatment.
Test pAtPCR2 will respond to cadmium
Our promoter pAtPCR2 is designed to respond to cadmium stress. In order to locate the complete promoter sequence from the AtPCR2 sequence, we amplified the first two thousand base pairs of the gene. We selected this specific range, as the promoter(s) of most genes would exist in the first two thousand base pair region. After locating the promoter, we construct sticky ends for the promoter to pair up with new vectors. This is accomplished by adding two different pairs homology arms to the 5’ end of the promoter sequence. One pair comes from the vector GFP (green fluorescent protein), and the other one comes from the vector GUS (beta-glucuronidase). After completion of homology pairs, the modified gene sequence is supposed to be activated and to generate bio-luminescence under cadmium stress.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 22
Illegal XbaI site found at 1249
Illegal SpeI site found at 1572 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 22
Illegal SpeI site found at 1572 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 22
Illegal XhoI site found at 477 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 22
Illegal XbaI site found at 1249
Illegal SpeI site found at 1572 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 22
Illegal XbaI site found at 1249
Illegal SpeI site found at 1572 - 1000COMPATIBLE WITH RFC[1000]
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