Part:BBa_K2448035
Psicose Biosensor pPsiA-At PsiR-At with D-Psicose 3-epimerase from Clostridium cellulolyticum
This part is the Psicose Biosensor BBa_K2448025 with a downstream D-Psicose 3-epimerase (Dpe) from Clostridium cellulolyticum (BBa_K2448021) under the control of the pTacI promoter (BBa_K864400).
Usage and Biology
Biosensors rely on a basic theoretical principle: a certain concentration of a molecule of interest induces the proportional production of an easily detectable output, like fluorescence. Transcription-factor based biosensors allow quick and cheap detection or quantification of various chemical compounds.
Psicose biosensors were the lynchpin of our iGEM project. In order to improve the enzymatic production of psicose, we needed an efficient method to screen large banks of mutants. To address this problem, we designed a collection of specific biosensors able to detect psicose concentration in vivo (BBa_K2448025, BBa_K2448026, BBa_K2448027, BBa_K2448028, BBa_K2448029, BBa_K2448030 an BBa_K2448031).
The best biososensor of this collection proved to be BBa_K2448025 and we decided to use it to screen a library of mutants and identify the cells carrying an improved version of our enzyme, the D-Psicose 3-epimerase (Dpe) from Clostridium cellulolyticum (BBa_K2448021).
This part is was constructed for this purpose. It contains the Psicose Biosensor BBa_K2448025 with a downstream D-Psicose 3-epimerase (Dpe) from Clostridium cellulolyticum (BBa_K2448021) under the control of the pTacI promoter (BBa_K864400). The general architecture of this part is illustrated in Figure 1.
Figure 1. General architecture of this part.
In addition, we build by error prone PCR a bank of mutant Dpe that we screened using the protocol presented in the Experience page.
Screening this library of mutants of our enzyme, the D-Psicose 3-epimerase (Dpe) from Clostridium cellulolyticum (BBa_K2448021) using this part (and it's mutant derivatives) allowed us to:
- show that our screening process works under realistic conditions. The biosensor we developed is able to give accurate assessment of the psicose production on positive control clones carrying the wild-type enzyme, but also on bacteria carrying mutant versions of Dpe. According to raw data, fluorescence of the positive control (WT, BBa_K2448035) is around 4500 arbitrary units. Biosensor characterization allowed us to correlate fluorescence and psicose concentration. We can estimate that psicose produced by the enzyme in WT is around 10 grams per liter which is coherent with measurements perform with BBa_K2448033.
- This screening process also allowed us to screen around 400 mutants (Figure 2) and discover at least 2 enzymes with potentially improved activity (>30%) according to the fluorescence data. Four other variants displayed potentially improved activity, but of less then 10%.
Figure 2. In vivo characterization of D-psicose-3-epimerase mutants using pPsiA-PsiR biosensor from Agrobacterium tumefaciens (BBa_K2448025) in E. coli DH5alpha. The total number of mutants screened were 367. All the data points are Fluorescence/ OD of each mutant normalized by the same value from controls (WT, BBa_K2448035). The graphs show the repartition of all the mutants function to their relative activity compared to WT (A) and the repartition of the mutant in range of activity (B).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1337
Illegal XhoI site found at 62
Illegal XhoI site found at 2618 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 104
Illegal AgeI site found at 791
Illegal AgeI site found at 2351
Illegal AgeI site found at 2463
Illegal AgeI site found at 3252 - 1000COMPATIBLE WITH RFC[1000]
| None |