Part:BBa_K2442108

AraC mutant library variant 4

This part contains LacI-regulated promoter R0011, weak RBS B0032 and a mutant variant of the AraC coding region. The protein no longer responds to L-arabinose. It is not responsive to the molecules tested by Glasgow 2017 iGEM team, but may be responsive to a compound not yet identified. 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 used for mutagenesis was obtained from BBa_I0500.

Usage and Biology

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 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.

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), mevalonate and triacetic acid lactone. 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. By following the protocol from Tang et al. (2008) 4 mutant AraC variants were obtained: BBa_K2442105, BBa_K2442106, BBa_K2442107, BBa_K2442108.

By following our protocol, future teams can generate AraC variants with altered effector specificity. This can serve as a toolkit to develop biosensors for detection of small molecules which do not bind to any receptor molecules in nature.

Characterization

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 was turned into a BioBrick and the part 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 NanoDrop^TM 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. PCR product was then purified and ligated into regulatory plasmid pSB1C3+R0011+B0032 to generate araC mutant library. Visit our [http://2017.igem.org/Team:Glasgow/araC wiki] for details.

Figure 2: Primer sequences used in library construction. Degenerate sequences are highlighted in magenta. N, any base. S, strong base (G or C). Red, random 6bp sequence (to allow restriction digest). Yellow, BioBrick prefix. Cyan, reverse compliment of BioBrick suffix.

Fluorescence-based screening

Plasmid DNA containing the araC mutant libraries were transformed into DS941 carrying the reporter plasmid BBa_K2442102. Transformants were plated on LB agar medium containing chloramphenicol plus kanamycin (to select for K2442102 and K2442104) plus one of the tested inducers: arabinose, xylose or decanal. Fluorescence images of the conditional transformation plates were obtained, and colonies which exhibited fluorescence were observed as dark colonies on the scan. 200 of those which appeared dark on xylose or decanal plates were re-streaked onto plates containing xylose, arabinose, decanal or no additive. Each colony of interest was picked and then immediately short-streaked onto each new condition plate. After overnight incubation at 37°C, fluorescence scans of each plate were again obtained using the scan fluorescent scanning method (Fig. 3). BBa_K2442105 (circled in green) was picked as it appeared to be fluorescing under decanal, xylose or arabinose conditions and less so when there was no additive in the agar.

Figure 3: Fluorescence scans of re-streaks of mutant DS941 colonies which appeared to show fluorescence under xylose or decanal conditions. Mutants which appeared to have novel behaviour are labelled 1-4. Mutant 1: BBa_K2442015; Mutant 2: BBa_K2442106; Mutant 3: BBa_K2442107; Mutant 4: BBa_K2442108

Liquid culture fluorescence assay

Colony of interest was then inoculated into L-broth containing chloramphenicol and kanamycin and grown overnight. The following day the culture was diluted 1:100 into fresh L-broth containing the above antibiotics plus one of the inducer conditions: 40mM xylose, 40mM arabinose, 2mM decanal. 200l of each culture to be tested was placed in a well of a clear-bases 6-well plate, then incubates at 37°C shaking at 300 rpm for 12 hours in a BMG Fluorostar Omega fluorescence plate reader. Fluorescence of each culture was measured at 1 hour time intervals during the culture experiment, using excitation wavelength 485nm and emission wavelength 530nm. Cell growth was simultaneously tracked by measuring optical density at 600 nm. Figure 4 compares average relative fluorescence over optical density at hour 8 between mutants and controls.

Results

Mutant 4, K244108, showed levels of fluorescence comparable to empty DS941 cells (Fig.4). There was no obvious difference between arabinose, decanal and no additive when liquid culture was examined. As the protein contains mutations only in its binding pocket, it has most likely lost its ability to bind or respond to L-arabinose. Although not found to be responsive to xylose or decanal, it is likely that this AraC variant is responsive to a molecule not yet identified. Due to time constraints we were unable to test for other compounds, and this should be done as a future experiment.

Sequencing result came back inconclusive due to mispriming. The sequencing is to be repeated.

Figure 4: 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 (araC^-. BioBrick numbers (K244210..) specified. All parts are in plasmid backbone pSB1C3. K2442102: reporter plasmid pBADmin+GFP. K2442104: regulatory plasmid R0011+B0032+AraC

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]