Difference between revisions of "Part:BBa K2442105"

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AraC mutant library variant 1

This part contains LacI-regulated promoter R0011, B0032 ribosome binding site and a mutant variant of the 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. Wild type araC was obtained from BBa_I0500.

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.

Figure 1: Crystal strycture of AraC binding pocket with bound L-arabinose (LA), showing key residues that play a role in ligand binding. Residues targeted for mutagenesis in our experiment are underlined in red. Water molecules are shown as spheres (adapted from Tang et al., 2008.)

The aim of our project was to mutagenize residues within the AraC ligand-binding pocket to generate mutants with altered effector specificity. Several previous studies have shown that AraC protein can be engineered to activate transcription in response to non-native small molecules. Some of them include D-arabinose (enantiomer of L-arabinose)^[1], mevalonate^[2] and triacetic acid lactone^[3]. Site-saturation mutagenesis of residues positioned within the ligand-binding pocket of AraC, coupled with fluorescence-based cell sorting, allowed the groups to isolate AraC variants with altered effector specificity. Fig. 1 represents key residues of AraC protein that play a role in ligand binding. Previous studies have shown that mutagenesis of amino acids in positions 8, 24, 80 and 82 is sufficient to change effector specificity^[4]. By following the protocol from Tang et al. (2008) 4 mutant AraC variants were obtained: BBa_K2442105, BBa_K2442106, BBa_K2442107, BBa_K2442108.

Library Construction

Primer sequences are listed in Fig. 2. araC gene was amplified from the BBa_I0500 part using primers araC_F and araC_R. The sequence is available as BBa_K2442103. The PCR product was then used in three PCR reactions with the following sets of primers: araC_Frag1_F and araC_Frag1_R; araC_Frag2_F and araC_Frag2_R; araC_Frag3_F and araC_Frag3_R. The PCR conditions were: 98°C for 30s, then 35 cycles of 98°C for 10s, 67.5°C (for F1 and F2) or 59.8°C (for F3) for 30s and 72°C for 1min, then final step of 72°C for 10 mins. The reactions resulted in 3 araC fragments (F1, F2 and F3). Primers araC_Frag1_F, araC_Frag1_R and araC_Frag2_R contain the degenerate sequences NNS at specific triplets so that fragments F1 and F2 contain saturation mutagenesis at codon positions 8, 24, 80 and 82. PCR products were gel purified and DNA concentration of each was measured with a NanoDropTM spectrophotometer. Equimolar aliquots (0.15pmol each) of adjacent fragments were combined (F1+F2 and F2+F3) and PCR-assembled without primers under the following conditions: 98°C for 30s, then 15 cycles of 98°C for 10s, 60°C for 1min and 72°C for 40s, then 72C for 10 mins. 15l of each reaction product was combined and PCR-assembled without primers under following conditions: 20 cycles of 98C for 30s and 72C for 40s. Finally primers araC_BBPre_F and araC_BBSuf_R were added and a final round of PCR was ran with the following programme: 98°C for 30s, then 30 cycles of 98°C for 10s, 60°C for 1 min and 72°C for 40 s, then 72°C for 10 min. QIAQuick PCR purification of the product was performed according to the Qiagen protocol.

Characterization

Figure 3: Average relative fluorescence over optical density at hour 8. Shows fluorescence levels under no additive, 40mM Arabinose, 40mM Xylose and 2mM decanal. The cells which the plasmids have been transformed into are DS941 or DH5a (specified). Biobricks (K244210..) and plasmids (pSB1C3, pSB3K3) specified.

Sequence and Features

1. ↑ Tang, S., Fazelinia, H., and Cirino, P. (2008). AraC Regulatory Protein Mutants with Altered Effector Specificity. Journal Of The American Chemical Society 130, 5267-5271.
2. ↑ Tang, S., and Cirino, P. (2010). Design and Application of a Mevalonate-Responsive Regulatory Protein. Angewandte Chemie 123, 1116-1118.
3. ↑ Frei, C., Wang, Z., Qian, S., Deutsch, S., Sutter, M., and Cirino, P. (2016). Analysis of amino acid substitutions in AraC variants that respond to triacetic acid lactone. Protein Science 25, 804-814.
4. ↑ Tang, S., Fazelinia, H., and Cirino, P. (2008). AraC Regulatory Protein Mutants with Altered Effector Specificity. Journal Of The American Chemical Society 130, 5267-5271.