Part:BBa_K2442104
lacI regulated promoter + B0032 RBS + AraC cds
This part contains LacI-regulated promoter R0011, weak RBS B0032 and AraC coding region. LacI-regulated promoter is derived from the lac operon and is inducible by IPTG. AraC is derived from the L-arabinose operon from Escherichia coli, and was obtained from BBa_I0500.
This BioBrick together with BBa_K2442101 is an improvement of BBa_I0500 by splitting its constituent parts - pBAD promoter and araC - into two separate plasmids to allow greater control over production of AraC. Placing araC under control of an inducible promoter instead of PC allows to induce expression of the protein only when required, and takes the translational load off growing host cells.
This part belongs to a collection of parts made by [http://2017.igem.org/Team:Glasgow Team Glasgow 2017]:
- BBa_K2442101: minimal pBAD promoter
- BBa_K2442102: reporter plasmid - minimal pBAD promoter + GFP
- BBa_K2442103: araC coding sequence
- BBa_K2442104: regulatory plasmid - araC coding sequence under control of LacI-regulated promoter
Usage and Biology
The L-arabinose operon is naturally found in Escherichia coli. The regulatory protein, AraC, acts as a dimer. The operon contains two regulatory cis elements: the PC promoter for the synthesis of AraC, and the pBAD promoter for synthesis of enzymes required for catabolism of L-arabinose. pBAD promoter contains 3 half sites that each bind to one subunit of AraC - O2, I1, I2. The O1 site is composed of O1L and O1R half sites, which bind both subunits. The O2 half site is within the araC coding region.
In absence of L-arabinose the AraC dimer binds to operator half-sites O2 and I1. This causes DNA looping upstream of the pBAD promoter which represses transcription by excluding RNA polymerase from binding to pBAD or PC. Binding of L-arabinose causes a conformational change in the protein such that the DNA-binding domains of the dimer bind to adjacent I1 and I2 half-sites, giving access for RNA polymerase and cyclic AMP receptor protein (CRP) to bind pBAD.
To improve BBa_I0500, we split the araC and pBAD into two separate parts (BBa_K2442101 and BBa_K2442104). araC coding sequence as described by Miyada et al (1980) [1] was amplified by PCR from BBa_I0500 using primers araC_BBPre_F and araC_BBSuf_R (Fig. 1). The primers were designed to incorporate BioBrick prefix and suffix to either side of araC to make the part BioBrick-compatible.
The aim of our project was to mutagenize 4 residues within the AraC ligand-binding pocket to generate mutants with altered effector specificity. araC coding region as in this part served as a template for PCR-based mutagenesis. We also placed this part downstream of LacI regulated promoter BBa_K2442104 to allow for greater control over production of AraC. For mutant screening we transformed araC-negative E. coli strain, DH5α, with regulatory plasmid carrying the araC mutant library and reporter plasmid BBa_K2442102. Mutants were tested for responsiveness to xylose, decanal, arabinose and no inducer based on fluorescence. BBa_K2442104 served the purpose of control, to check whether WT AraC functions as expected.
BBa_K2442104 and BBa_K2442101 can be used together to construct arabinose-inducible systems. AraC from BBa_K2442103 can be placed under control of other promoters of choice, including inducible promoters that allow for greater control over protein production.
Characterization
To test function of AraC, we transformed araC-negative E. coli strain DS941 with our reporter plasmid BBa_K2442102 (pBAD+GFP pSB3k3) and regulatory plasmid BBa_K2442104 (R0011+B0032+araC pSB1C3). When plated on arabinose, all cells showed fluorescence showing that AraC is fully functional (i.e. activates pBAD in presence of arabinose, but not other sugars) (Fig. 2). The cells showed fluorescence even without the addition of IPTG, showing that LacI-regulated promoter R0011 is leaky – background transcription of araC from BBa_K2442104 is sufficient to activate pBAD.
We next measured fluorescence of cells transformed with BBa_K2442104 under conditions of arabinose, xylose, decanal and no inducer (Fig. 3). E. coli strain DH5α (araC-positive) transformed with BBa_K2442104 served as control to show that BBa_K2442104 on its own does not increase fluorescence. Cells transformed with reporter plasmid on its own showed no fluorescence, showing that pBAD is not leaky – requires AraC for activation. DS941 cells carrying BBa_K2442104 and reporter plasmid showed high fluorescence in presence of arabinose, and no fluorescence at other conditions. This showed that AraC activates pBAD only in presence of arabinose, which makes it highly specific. DH5α carrying reporter plasmid alone showed comparable levels of fluorescence, showing that chromosomal copy of araC induces pBAD from a low copy number plasmid pSB3k3 at equal levels to when expressed from pSB1C3.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
- ↑ Miyada, C., Horwitz, A., Cass, L., Timko, J., and Wilcox, G. (1980). DNA sequence of the araC regulatory gene from Escherichia coli B/r. Nucleic Acids Research 8, 5267-5274.
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