Difference between revisions of "Part:BBa K833012"

Latest revision as of 03:58, 4 October 2012

Lactate sensor derived from the MglB protein from E. coli

This part is a periplasmic lactate sensor derived from site directed mutagenesis of the E. coli MglB protein to create the lactate sensor described by Looger et al. (Looger, LL. et al., Computational design of receptor and sensor proteins with novel functions (Nature 2003). The MglB gene was mutated to a produce a protein with amino acid changes at the sites reported by Looger et al., (Y10K, D14K, N91K, K92L, H152M, D154H, R158K, W183K, D236A and N256D) by proper primer design and use of the Strategene Multi Site-directed Mutagenesis Kit and protocol. **Note that the amino acid numbers referred to come from the Nature paper by Looger et all, however they made a truncation of the protein so for the actual amino acid numbers of the full length protein add 21 amino acids. Due to the multiple mutations, two rounds of mutagenesis were necessary to incorporate all mutations, and several colonies had to be sequenced after each round in order to identify colonies which contained all of the mutations.

This sensor interacts with the Trz fusion protein (iGEM part Bba_J58009) when it binds lactate. The Kd (2 uM) is within the physiological range of lactate concentrations for many cell growth conditions. Trz is a Trg-EnvZ fusion which phosphorylates OmpC when activated. A GFP-based OmpC reporter is available in the iGEM registry (iGEM part BBa_K116503). The reporter we are using is a lacZ reporter, which gives a 4X induction with 100uM L-lactate.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 982
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 982
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 982
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 982
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 982
1000
COMPATIBLE WITH RFC[1000]

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 __NOTOC__
 <partinfo>BBa_K833012 short</partinfo>
-This part is a lactate sensor derived from site directed mutagenesis of the E. coli MglB protein.
+This part is a periplasmic lactate sensor derived from site directed mutagenesis of the E. coli MglB protein to create the lactate sensor described by Looger et al. (Looger, LL. et al., Computational design of receptor and sensor proteins with novel functions (Nature 2003).   The MglB gene was mutated to a produce a protein with amino acid changes at the   sites reported by Looger et al., (Y10K, D14K, N91K, K92L, H152M, D154H, R158K, W183K, D236A and N256D) by proper primer design and use of the Strategene Multi Site-directed Mutagenesis Kit and protocol. **Note that the amino acid numbers referred to come from the Nature paper by Looger et all, however they made a truncation of the protein so for the actual amino acid numbers of the full length protein add 21 amino acids. Due to the multiple mutations, two rounds of mutagenesis were necessary to incorporate all mutations, and several colonies had to be sequenced after each round in order to identify colonies which contained all of the mutations.
+This sensor interacts with the Trz fusion protein (iGEM part Bba_J58009) when it binds lactate.  The Kd (2 uM) is within the physiological range of lactate concentrations for many cell growth conditions.  Trz is a Trg-EnvZ fusion which phosphorylates OmpC when activated.  A GFP-based  OmpC reporter is available in the iGEM registry (iGEM part BBa_K116503).   The reporter we are using is a lacZ reporter, which gives a 4X induction with 100uM L-lactate.
 <!-- Add more about the biology of this part here