Part:BBa_K763001
pGroE + Gene encoding AsRed2
This construction is made up of three parts:
1. The transcription factor-binding site inside the promoter
2. the coding sequence, which contains a synthetic fluorescent (red) protein. Our construction uses the well-known high chaperon expression when the bacterium is suffering from heat shock [1]. We have taken the promoter sequence of groE, one of these proteins highly produced when heat shock [2]. When is the protein synthesized? In order to obtain the red fluorescent protein a condition has to be met. High temperatures for E. coli must be reached, like for example from 42ºC degrees. We focused on 44ºC because here we got the best results.
The molecular mechanism underlying this phenomenon is as follows: high temperatures are sometimes harmful for microorganisms, so they have adapted successfully for such a strong condition by synthesizing some proteins, called HSP (Heat Shock Proteins), that help in protein folding. Heat-shock response is mediated by the sigma32 factor [3]. This alternative sigma factor allows the RNA polymerase to bind to some consensus promoter sequence. groE has this sequence in its promoter [4]. That is because this gene encodes a chaperon protein, GroE. Chaperone proteins are a group of proteins present in all cells; many of them are heat shock proteins, whose function is to assist the folding of other proteins in the newly formed protein synthesis.
In the case of GroE, it processes a nonnative polypeptide in a cycle consisting of three steps. First, the polypeptide substrate is captured by GroEL. Upon binding of the co-chaperone GroES and ATP, the substrate is then discharged into a unique microenvironment inside of the chaperone, which promotes proper folding. After hydrolysis of ATP, the polypeptide is released into solution. Moreover, GroE may actively increase the folding efficiency, e.g. by unfolding of misfolded protein molecules. This chaperon has an important role in heat shock process too, helping other proteins not to denature.
How did we deal with this construction? We were able to activate the promoter after a heat shock, like keeping the culture at 44ºC during 5 min. However, since we wanted a very good modeling for this construction, we developed a lot of experiments with different temperatures, different times of incubation, as well as gradating the temperature ranging from 37ºC to 46ºC.
Caracteritation
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 507
- 1000COMPATIBLE WITH RFC[1000]
| None |