Difference between revisions of "Part:BBa K1893018"

Latest revision as of 19:55, 28 October 2016

Arabinose inducible 3-isopropylmalate dehydrogenase (pBAD+leuB)

This composite part consists of a leuB coding sequence under the control of an arabinose-inducible promoter (BBa_K1893015). leuB encodes for 3-isopropylmalate dehydrogenase, an enzyme in the leucine biosynthesis pathway that catalyses the oxidation of 3-isopropylmalate to 3-carboxy-4-methyl-2-oxopentanoate.

Usage and Biology

We initially considered auxotrophy as a method of controlling growth through our growth-regulation module. The pBAD+leuB was designed to investigate the effect of leuB suppression on the growth of E. coli leuB knockout strains. The knockout strains were to be grown in media lacking leucine, so that only cells expressing leuB would be capable of growth. Once the effect of leuB expression on growth in starvation conditions had been determined, we would then place leuB under the control of an inverter. As a result, our STAR part could activate expression of the inverter, which would suppress leuB expression and lead to growth inhibition.

However, introducing an inverter into the system would have greatly increased the complexity of our circuit. Furthermore, we realized that having to generate leuB knockout strains and media lacking leucine contradicted the design specifications of our system, which aimed to make the implementation of co-culture experiments as simple as possible. As a result, we decided against further pursuing leuB as a method of growth control.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 1342
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 1281
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 1116
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI site found at 1098

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K1893018 short</partinfo>
-The leucine biosynthesis gene placed under control of a pBAD promoter, used to control growth of an E. coli leucine auxotroph using arabinose.
+This composite part consists of a leuB coding sequence under the control of an arabinose-inducible promoter [https://parts.igem.org/Part:BBa_K1893015 (BBa_K1893015)]. leuB encodes for 3-isopropylmalate dehydrogenase, an enzyme in the leucine biosynthesis pathway that catalyses the oxidation of 3-isopropylmalate to 3-carboxy-4-methyl-2-oxopentanoate.
 ===Usage and Biology===
-The leucine biosynthesis pathway is activated in conditions of leucine starvation and can be manipulated for the purposes of auxotrophy experiments. We initially considered auxotrophy as a method of controlling growth through our growth-regulation module. This part was designed to investigate the effect of leuB suppression on the growth of E. coli leuB knockout strains. The knockout strains were to be grown in minimal media lacking leucine, so that only cells expressing leuB at a sufficient level would be capable of growth. Due to time constraints, and the difficulty of creating knockout strains, we were unable to obtain any characterisation data.
+We initially considered auxotrophy as a method of controlling growth through our growth-regulation module. The pBAD+leuB was designed to investigate the effect of leuB suppression on the growth of <i>E. coli</i> leuB knockout strains. The knockout strains were to be grown in media lacking leucine, so that only cells expressing leuB would be capable of growth. Once the effect of leuB expression on growth in starvation conditions had been determined, we would then place leuB under the control of an inverter. As a result, our STAR part could activate expression of the inverter, which would suppress leuB expression and lead to growth inhibition.
+However, introducing an inverter into the system would have greatly increased the complexity of our circuit. Furthermore, we realized that having to generate leuB knockout strains and media lacking leucine contradicted the design specifications of our system, which aimed to make the implementation of co-culture experiments as simple as possible. As a result, we decided against further pursuing leuB as a method of growth control.