Overview
In the design of our Campylobacter biosensor we decided to employ a genetic logic circuit. This would require the presence of two transcriptional input signals from Campylobacter-associated molecules before an output signal in the form of GFP fluorescence could be produced. We aimed to build and characterise two genetic AND-gates; one based on a split-GFP fluorescence system; and the other based on a splitting the Enterobacteria phage T7 RNA polymerase. We wished to quantify the responsiveness of both AND-gates with well-characterised small-molecule regulated promoters from the iGEM registry, and then proceed to utilising our own Campylobacter sensing transcriptional systems to drive the AND-gate biosensor. Both split-GFP and split-T7 RNAP gates were constructed from BioBrick parts and tested, however DNA sequence errors appear to have prevented the proper function of either system.
Introduction
After brainstorming our Campylobacter biosensor project idea, we began investigating any molecules that are present within - or secreted by – Campylobacter. In an ideal scenario, we would detect a small molecule that is associated with gastroenteritis-causing Campylobacter only - not in any other non-pathogenic subspecies of the Campylobacter genus – nor present in any other bacteria that might be inhabit a poultry-carcass environment. Additionally, in this ideal scenario, a well-characterised E. coli gene regulatory system with a strong output expression level would already exist.
Sensation
We identified two molecules of interest from Campylobacter for detection. One was xylulose, a 5-carbon ketopentose sugar. The second was the quorum sensing molecule autoinducer-2. Xylulose is a rare sugar most commonly associated with the pentosephosphate pathway, where xylulose-5-phosphate is an intermediate step. Interestingly, xylulose was found in the polysaccharide capsule of Campylobacter jejuni strain RM1221 (Gilbert et al., 2007). The presence of xylulose is not common in bacterial polysaccharide capsules, and the glyosidic bonds which incorporate xylulose were found to be extremely acid-labile. One sub-project aimed to exploit a xylulose-associated transcriptional activator system (mtlR-mtlE) from Pseudomonas fluorescens (mtlR). Another subproject aimed to mutagenize the well-characterised arabinose transcriptional regulatory system (araC-pBAD) from E. coli and modify its ligand specificity to the closely related structure of xylulose (araC). Our engineering subproject designed a functional prototype biosensor device that simplified a process of dissolving a swabbed input sample, processing the solution through acid and high temperature to release xylulose, and then delivering the sample to a waiting strain of genetically modified bacterial carrying the biosensor circuit for detection (hardware, applied design). Finally, purified xylulose is extremely expensive to purchase commercially, so for the testing of our biosensor we attempted to biosynthesise and purify the sugar ourselves (biosynthesis). The other molecule we identified as a marker for Campylobacter was autoinducer-2 (AI-2), a secreted quorum sensing molecule (quorum). AI-2 is a significantly less specific biomarker, as many varied gram-positive and gram-negative bacterial species sense their population density and surrounding bacterial environment using this molecule (Miller and Bassler, 2001). On the other hand, this ubiquity meant that AI-2 gene regulation was well characterised, with prior iGEM teams having worked on the natural E. coli AI-2 quorum sensing regulatory system.
The AND-gate
We hypothesised that building a combination biosensor requiring the input of two Campylobacter-associated molecules would reduce the risk of false-positives. To do this we envisaged a biosensor genetic circuit in the form of an AND-gate. In electronic circuitry, an AND-gate is a form of switch that only activates an output in the presence of two positive input signals (Figure 1).
SPACE FOR FIGURE 1
A genetic AND-gate uses transcriptional signalling in place of electricity. In the simplest form, we can design a genetic circuit where two gene products must be actively transcribed to produce an output signal. The interaction between the input and output signals can be direct or indirect, and we aim to test both types in this subproject.
The directly interacting genetic AND-gate we aim to test is gained by GFP into two individual protein subunits, either of which expressed alone would not be fluorescent. If both GFP subunits were to be transcriptionally controlled from separate promoters within the same cell, then an AND-gate would be formed. Only expression of both subunits at the same time would make the cells fluorescent. To split GFP we attempted to improve parts designed by a previous iGEM team, Davidson Missouri 2007, who split the GFPmut3b part E0040 into a 151 aa N-terminal fragment (named GFP1: I715019) and an 83 aa C-terminal fragment (named GFP2: I715020). These parts were designed for fusion to other proteins for Bimolecular fluorescence complementation (BiFC) assays for validation of protein-protein interactions. As such, the N-terminal part lacked a stop codon, and the C-terminal part lacked a start codon. The 2015 team NUDT_CHINA altered the GFP1 part to include a stop codon, now labelled K1789003, but we would have to design a PCR amplification to add a start codon to the GFP2 part for our uses.
For an indirectly interacting genetic AND-gate the inputs do not themselves act together as the output. The indirect gate we planned can be generated by splitting the RNA polymerase (RNAP) protein from Enterobacteria phage T7. T7 RNAP is a single subunit RNA polymerase that is most commonly used for strictly-controlled recombinant protein expression in E. coli. T7 RNAP begins transcription only from its cognate pT7 promoter sequence, and has complete orthogonality (no cross-reactivity) with E. coli promoter sequences. In addition, the T7 RNAP produces extremely reactive gene expression responses. Three hours following induction of T7 RNAP expression, a gene product expressed from the T7 promoter (pT7) may represent 50% of the cell’s mass (Studier and Moffatt, 1986). Interestingly, a proportion of the purified T7 RNAP protein is often found cleaved into two subunits, one small (179 aa), and one large subunit (701 aa). If these two cleavage products are expressed as separate polypeptides within a cell then they are capable of driving gene expression from a pT7 promoter, although at a moderately lower efficiency than the wild-type single polypeptide protein (Shis and Bennett, 2013). A Split T7 RNAP genetic circuit represents an indirect AND-gate when each subunit is controlled by a separate transcriptional activator and the pT7 promoter drives expression of an output such a GFP. No previous iGEM team has attempted to split and test a T7 RNAP AND-gate, although many have utilised pT7 (K1321338) and T7 RNAP (I716103) for protein over-expression.
We wished to build constructs of both split-GFP and split-T7 RNAP AND-gates by controlling the expression of split subunits from well-characterised orthogonal small-molecule regulated promoters within E. coli. Figure 2 shows a genetic diagram of our plan for testing the two different AND-gate circuits. It was an aim to quantify the difference in output GFP fluorescence level between the directly interactive split-GFP AND-gate, and the indirect T7-gate. If time was to permit, then testing would proceed to swapping input control of the gates over to the xylulose and autoinducer-2 regulated systems. Herein we will refer to each promoter + split protein construct as a “module”, i.e. a full AND-gate will require the presence and activation of both relevant modules.
SPACE FOR FIGURE 2
Methods
Standard molecular biology techniques. During this work, a standardised set for routine laboratory techniques were used such as restriction digest, gel electrophoresis, preparation of chemically competent E. coli, transformation of chemically competent E. coli, ligation reactions, oligonucleotide annealing, and PCR. These can be found on our protocols page. All ligation reactions described herein were transformed into NEB5α commercial chemically competent E. coli cells, plated on L-agar containing the required antibiotics, and grown at 37°C overnight.
E. coli strains.
SPACE FOR TABLE 1
PCR design. Split T7 RNA polymerase: PCR primers were designed to split T7 RNAP into two separate Biobrick compatible parts designed according to Shis and Bennett (2013). In their work, the 880 amino acid protein was split into 179 aa N-terminal, and a 701 aa C-terminal fragments. Forward and reverse primers to generate these two fragments were designed using Primer3 (Untergasser et al., 2012), aiming for primer annealing temperatures of roughly 60°C, and are shown in Table 2.
SPACE FOR TABLE 2
Primer design included the insertion of the weak strength ribosome binding site (RBS) B0032 upstream of the start codon of the two T7 RNAP fragments (green) highlighted sequence above. The addition of these RBS emulated the effect of Biobrick assembly by inclusion of the TACTAG assembly scar between the B0032 and RNAP fragment sequences (orange). Both fragment reverse primers were designed to result in the parts ending with double TAA stop codons, in the case of the N-term part this meant the insertion of the two stops, while the C-terminal part primer added one in addition to the natural TAA (red). The C-terminal F-primer also included a new start codon (purple). Forward primers included Biobrick prefixes and reverse primers included the Biobrick suffixes respectively, highlighted in yellow. All primers included a 6-bp sequence of DNA (blue) to allow efficient restriction digest of the outermost restriction enzymes, according to NEB instructions. The T7 RNAP fragments were amplified by a colony PCR of E. coli BL21<DE3>, a strain of E. coli that has the T7 RNAP integrated into its chromosome. A single colony of BL21<DE3> was picked from a streaked agar plate, then resuspended into 200 μl of TE buffer. 1 μl of this colony template was used in 50 μl NEB Phusion polymerase reactions, following their protocol, except with an extended (10 min) initial 95°C denaturation step to release genomic DNA from the colony template.
Split GFPmut3b (E0040). We designed a PCR reaction to generate a C-terminal GFP2 part which would work alongside the existing N-terminal GFP1 part K1789003 by NUDT_CHINA. Primer3 was used to design roughly 60°C Tm primers to amplify the coding sequence of the 83 aa C-terminal fragment, as shown in Table 3.
SPACE FOR TABLE 3
The GFP2 part was designed without the addition of a RBS. Only a start codon (purple) was inserted into the part sequence. Biobrick prefix/suffix (yellow), and flanking DNA (blue) are included in the fragment amplification. A 1 in 1000 dilution of miniprep DNA of the full GFP part E0040 was used as template for an NEB Phusion polymerase reaction, following their PCR condition protocol.
SDS-PAGE SDS-PAGE was performed using Bio-Rad 4–15% Mini-PROTEAN® TGX™ 10-well Precast Protein Gels, run in 1x Tris-Glycine buffer. E. coli GFP expression samples were prepared by recording the optical density of 1 ml samples of cells during culture, at 600 nm using a spectrophotometer. The 1 ml cell samples were transferred to separate microcentrifuge tubes and pelleted at 16,000 g for 5 minutes. Supernatant was discarded, and the cell pellets were resuspended in 2x final concentration Laemmli sample buffer (65.8 mM Tris-HCl, pH 6.8, 26.3% (w/v) glycerol, 2.1% SDS, 0.01% bromophenol blue, 20 mM DTT). Volume of sample-buffer to be added was normalised to OD values, where the highest OD sample received 100 μl of buffer, and lower ODs received less proportionally. Samples were boiled at 95°C for 5 minutes, then microcentrifuged at 16,000 g for 5 minutes. 20 μl of prepared samples were loaded into the pre-cast gel wells. Empty wells were filled with sample buffer. Gel-electrophoresis was performed at 150 V for roughly 45 minutes, or until the dye front reached the base of the gel. The gel was stained in Coomassie Blue (0.1% Coomassie Brilliant Blue G250, 50% methanol, 10% acetic acid) for 1 hour, then de-stained overnight in 10% methanol 10% acetic acid solution.
Results
Plasmid assembly. All plasmids were assembled using Biobrick assembly techniques, and a full list of parts used in this subproject is described below in Table 4. All parts are assembled the standard Biobrick vector pSB1C3 unless otherwise stated.
References
