Difference between revisions of "Team:Glasgow/araC"

Overview

Campylobacter jejuni is the leading cause of food poisoning in the UK, and is commonly found on uncooked poultry. Our team aims to develop a functional biosensor to detect Campylobacter jejuni on contaminated surfaces. We aim to engineer E.coli to produce GFP in response to the presence of xylulose – a sugar found in the outer capsule of C. jejuni. This report focuses on utilization of the L-arabinose sensing AraC regulatory protein and its regulated P_BAD promoter to construct one of the parts of the final detection system. The regulatory protein AraC binds to operator sequences in the P_BAD promoter and activates transcription of downstream genes only when bound to its substrate sugar (L-arabinose). Here we performed saturation mutagenesis on the araC gene to construct AraC mutant library with four randomized ligand-binding pocket amino acid residues corresponding to positions 8, 24, 80 and 82. The AraC mutant library was subjected to fluorescence-based screening using a GFP reporter. Due to unavailability of xylulose we tested for mutants responsive to xylose, decanal or no effector. We isolated and sequenced 4 mutants which showed altered effector specificity. Our results demonstrate an approach that allows to engineer a regulatory protein to respond to an effector molecule of interest and represent steps towards developing a functional biosensor to detect C. jejuni on contaminated surfaces.

Background

Campylobacter jejuni is a bacterial pathogen found in animal gastrointestinal tract, and is mostly associated with chickens. It is responsible for a vast number of food poisoning cases around the world. Current detection systems for the pathogen are time-consuming, expensive and inaccessible for everyday users. Our team aims to develop a functional biosensor by engineering Escherichia coli to detect xylulose - a sugar naturally present in the outer capsule of Campylobacter jejuni ^[1]. The sugar can be easily released from the capsule by contact with acid. Our team aims to construct two alternative regulatory systems as potential components of the biosensor. One of them exploits the MtlR operon, naturally found in Pseudomonas fluorescens. MtlR protein activates transcription of downstream genes upon binding to xylulose. Parts of the operon can be transformed into E. coli and utilized in such a way that in contact with xylulose, MtlR protein activates transcription of a gene producing a detectable signal, such as fluorescence from GFP. However, this approach may have some disadvantages. Coming from a different organism, the MtlR protein may not function in E. coli cells as expected. Furthermore, the MtlR operon has not been previously exploited in biosensors and may not show sufficient specificity to xylulose. Hence, we aim to construct an alternative regulatory system which exploits the L-arabinose operon.

Figure 1: Genes required for catabolism of L-arabinose and the operon components. araC expression is regulated by the PC promoter and is transcribed in the opposite direction from araBAD genes, which are controlled by the P_BAD promoter. AraC acts negatively (-) at the PC promoter and negatively or positively (+/-) at the P_BAD promoter. Adapted from Shleif (2000) ^[2]

The L-arabinose operon is naturally found in Escherichia coli. The regulatory protein, AraC, acts as a dimer. As depicted in Fig. 1, the operon contains two regulatory cis elements: the PC promoter for the synthesis of AraC, and the P_BAD promoter for synthesis of enzymes required for catabolism of L-arabinose. The mechanism of the operon regulation has been described by Schleif et al (2000) and is summarized in Fig. 2. The P_BAD 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 P_BAD promoter which represses transcription by excluding RNA polymerase from binding to P_BAD 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 P_BAD. This results in transcriptional activation.

Figure 2: Regulation of the L-arabinose operon by arabinose. In the absence of L-arabinose, AraC dimer binds to O₂ and I₁ half sites causing DNA looping, which prevents RNA polymerase from accessing P_BAD. Upon binding of L–arabinose, the AraC dimer binds I₁ and I₂ half sites instead, allowing transcription of the polycistronic araBAD mRNA. araC is transcribed in opposite direction from araBAD, and is under control of the PC promoter. RNA polymerase and AraC compete for binding at O₁ and PC. Adapted from Shleif (2000) ^[3]

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)^[4], mevalonate^[5] and triacetic acid lactone^[6]. 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. Based on these findings we aimed to use multiple site-saturation mutagenesis and fluorescence-based screening to generate mutant AraC responsive specifically to xylulose. Although xylulose comes in the form of two enantiomers, we chose to focus on D-xylulose, which is the isomer present in the capsule of C. jejuni (Gilbert et al, 2007). Both L-arabinose and D-xylulose are monosaccharides with five carbons, with molecular formulas C5H10O5. As seen in Fig. 3a, arabinose is an aldopentose with an aldehyde functional group at carbon position 1, while xylulose is a ketopentose, possessing a ketone group instead. The 3-dimensional structure of D-xylulose differs from that of L-arabinose, with the most pronounced difference being the 5-membered vs 6-membered heterocyclic ring in its structure. Fig. 3b 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^[7].

Figure 3: (a) Structures of L-arabinose and D-xylulose. (b) 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 are underlined in red. Water molecules are shown as spheres (adapted from Tange et al., 2008.)

Aims

• Aim 1

To split the AraC regulatory system into two plasmids: regulatory plasmid expressing AraC and AraC mutants and reporter plasmid expressing GFP under control of PBAD promoter.

• Aim 2

To perform multiple site-saturation mutagenesis on the AraC protein at residue positions 8, 24, 80 and 82.

• Aim 3

To isolate AraC variants with altered effector specificity using fluorescence-based screening.

Materials and Methods

Plasmid design

Library Construction

Plasmid construction was as follows.

Oligos containing parts R0011 (LacI-regulated promoter) upstream of B0032 (ribosome-binding site) were synthesised by Integrated DNA Technologies (IDT). Each strand had different, incomplete prefix and suffix sequences, such that when annealed the resulting dsDNA had BioBrick-compatible sticky ends for ligation into EcoRI and PstI digested BioBrick vectors. The resulting product was ligated into high copy number pSB1C3 vector digested with EcoRI and PstI. The ligation reaction was introduced into chemically competent E.coli strain DH5, and sequence from a chosen colony was verified.

WT araC was amplified from BBa_I0500 using primers araC_BBPre_F and araC_BBSuf_R (see Table 1) to introduce BioBrick prefix and suffix to both ends of WT araC. The PCR product was subsequently digested with XbaI and PstI. pSB1C3-R0011-B0032 was digested with SpeI and PstI. The two digested parts were ligated to generate K2442104 plasmid with WT araC. Sequence was verified.

P_BADmin was synthesised by Integrated DNA Technologies (IDT) as a G-block and digested with EcoRI and PstI. The cut product was then ligated into pSB3k3 vector digested with EcoRI and PstI. The ligation product was introduced into E.coli strain DH5 by chemical transformation and the sequence was verified. Part I3500 was then digested with enzymes XbaI and PstI and ligated into pSB3k3-P_BADmin, digested with SpeI and PstI. This resulted in the final reporter plasmid K2442102, and the sequence was verified.

Fluorescence-based screening

Liquid culture fluorescence

Results and Discussion

Outlook

References