Difference between revisions of "Part:BBa K2442103"

Revision as of 16:06, 30 October 2017

E. coli AraC coding region

This part contains AraC coding region. AraC is derived from the L-arabinose operon from Escherichia coli, and was obtained from BBa_I0500.

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 O_1L and O_1R 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.

Figure 1: Primers used to amplify araC from BBa_I0500 by PCR. Forward: araC_BBPre_F. Reverse: araC_BBSuf_R. The primers were designed to incorporate BioBrick prefix (yellow) and suffix (reverse compliment in cyan). Melting temperature (Tm) specified. Random 6bp long DNA sequence (red) was added at the beginning of each primer to allow for efficient restriction digest.

Figure 1: Primers used to amplify araC from BBa_I0500 by PCR. Forward: araC_BBPre_F. Reverse: araC_BBSuf_R. The primers were designed to incorporate BioBrick prefix (yellow) and suffix (reverse compliment in cyan). Melting temperature (Tm) specified. Random 6bp long DNA sequence (red) was added at the beginning of each primer to allow for efficient restriction digest.

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 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.

"araC" can be placed under control of any inducible promoter of choice and together with BBa_K2442101 or BBa_K2442102 can be used together to construct arabinose-inducible systems.

By following our protocol, "araC" can serve as a template for PCR-based mutagenesis to generate AraC variants with altered effector specificity. This could be used by other teams to develop biosensors for detection of small molecules which do not bind to any receptor molecules in nature.

Characterization

To test function of AraC, we transformed araC-negative E. coli strain DS941 with our reporter plasmid K2442102 (pBAD+GFP pSB3k3) and K2442104 (R0011+B0032+araC). When plated on arabinose, all cells showed fluorescence showing that AraC is fully functional (activates pBAD in presence of arabinose, but not other sugars).

Figure 2: Fluorescence scan of DS941 cells with both reporter and regulatory plasmids incorporated. Cells are plated on nutrient agar supplemented with 40mM arabinose.

We next measured fluorescence of cells transformed with K2442104 under conditions of arabinose, xylose, decanal and no inducer (Fig. 3). E. coli strain DH5α (araC-positive) transformed with K2442104 served as control for basal levels of 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 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.

Figure 1:Average relative fluorescence over optical density at hour 8. Shows fluorescence levels under: no additive, 40mM Arabinose, 40mM Xylose and 2mM decanal. The E. coli strains which the plasmids have been transformed into are DS941 or DH5α (specified). BioBricks (K244210..) and plasmids (pSB1C3, pSB3K3) specified.

Sequence and Features

1. ↑ 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.