Part:BBa_K2940000
Zinc-sensitive Biosensor producing CotA Laccase
A simple device for the production of laccase in response to the presence of zinc in the culture medium. The device contains the cotA laccase and smtB metallothionein repressor genes and the smtA promoter sequence which contains the binding motif for SmtB. The SmtB protein is a repressor; in the absence of any inhibitory molecule, SmtB will bind to and repress transcription from the PsmtA promoter. The sensor is constructed with two copies of PsmtA, one for each gene, so that transcription from both genes is controlled by the same inducer, giving the entire sensor binary ‘on’ and ‘off’ states. This was intended to reduce metabolic load and increase the sensor’s specificity. SmtB is usually produced constitutively, however, by reducing the overall amount of SmtB present in the ‘off’ state, it was thought that this would decrease any inappropriate repression when metals were present, while still allowing repression to be maintained. It was also thought that increasing the SmtB production when repression is relieved would shorten the time taken for the sensor to switch back to the ‘off’ state when metal was no longer present. This would, in theory, result in a much more reliable sensor.
Usage and Biology
The repressor gene chosen here is smtB, a member of the SmtB-ArsR family of metal-sensing proteins. SmtB is a small dimeric protein that binds to an inverted repeat sequence present in the promoter of smtA, a metallothionein protein involved in heavy metal homeostasis [11]. This was chosen as it has already been used to create a zinc-inducible expression system in Synechococcus [10].
CotA laccase is a well-characterised classical laccase enzyme from Bacillus subtilis. It was chosen as previous iGEM teams have worked with this enzyme and there is significant literature to support its ability to degrade a variety of azo dyes. This sequence was taken from the B. subtilis genome sequence (GenBank ID: GU972589). A single base pair substitution was made to remove a PstI restriction site in the coding sequence.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1988
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 327
Illegal BamHI site found at 1745 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 665
Illegal NgoMIV site found at 1559
Illegal AgeI site found at 199
Illegal AgeI site found at 362 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 1017
Experimental Data
Zinc
Once the biosensor was constructed, testing was carried out to see if it was capable of responding to induction from zinc and producing laccase. An ABTS assay using whole cells was carried out to determine if there was appreciable laccase activity from the biosensor in response to a variety of concentrations of zinc sulphate heptahydrate (ZnSO4.7H2O) ranging from 1 to 800µM. In the wild type S. elongatus, SmtB response to zinc has been observed at concentrations as low as 4µM [4], while the concentration of zinc in a typical textile effluent may be between 7µM (0.51 mg/l) [1] and 91µM (6mg/l) [5]. In general, there is some initial laccase activity seen even in the uninduced samples, which is expected due to the nature of the promoter-repressor interaction. In this set of results, laccase activity is always significantly greater in the biosensor cells than in either control sample, suggesting that the biosensor is indeed functional and producing laccase. Additionally, a small but significant induction by zinc can be seen between the 1µM and 4µM concentrations (p=0.0481). However, as the zinc concentration increases above this, there is a marked drop in observed laccase activity in all samples. At the highest concentrations the activity observed, while still significantly higher than in the control samples, is very low overall. It was theorised that this could either be due to zinc toxicity reducing the amount of laccase in the sample, or due to the metal interfering with the laccase’s ability to oxidise the substrate.
SDS-PAGE was performed to visualise whether or not the laccase was being produced at all concentrations, and where it was present. A previous iGEM team had noted a large increase in laccase activity in supernatant using the PelB peptide [41], so it was important to check where the laccase was being secreted.
From SDS-PAGE, it became very clear that the laccase was not being secreted into the supernatant, despite the results obtained by the previous team. However, there did seem to be a faint laccase band present in the pellet samples, which was reasoning behind testing the biosensor using cell lysate rather than whole-cell culture, in order to release the laccase from the cell.
Cobalt and Nickel
Cobalt and nickel are both commonly found in textile wastewater [1,6] and are also known to be sensed by members of the SmtB-ArsR family [7,8]. In order to test if the sensor showed any cross-activation by these metals, it was induced in the same manner as zinc and both laccase activity assays and SDS-PAGE were performed.
For both cobalt and nickel, the results are considerably less clear than for zinc. While for zinc, laccase activity is always significantly higher than both controls, this is not always the case for the other two metals. While in some cases there is a clear change for the biosensor, such as the decrease in activity from no cobalt to 1µM, this drop is not observed in the controls, which stay approximately the same, or increase. However, the same trend of laccase activity sharply decreasing at higher concentrations is present in all three metals tests, suggesting that all three are toxic at high levels. A band at the correct size of laccase was observed at all concentrations in the cobalt-induced samples but was only clearly present at 15µM and 50µM in the nickel-induced samples.
Issues with Construction
Upon sequencing the construct, we found that during cloning, a recombination event had occurred that had not previously been noticed. This replaced what was intended to be the second copy of the PsmtA sequence, controlling cotA expression, with a random portion of E. coli genomic sequence. It is thought that this may be why the laccase activity observed is quite low.
The second copy of the promoter appeared to be completely wrong in the sequence data. Nucleotide BLAST of the 95bp sequence which appears instead of PsmtA, shown in Figure 8, shows a 100% homology to the E. coli K12 genome sequence coding for a putative ATP binding protein (GenBank CP027060.1, Region: 3284213..3284307) [9].
There also appears to be a frameshift mutation in the smtB sequence in this sequencing data, caused by a single base pair deletion, shown in Figure 9. In this case, it is unclear whether the frameshift is present in the original synthesised DNA or has been introduced at a later step. The frameshift observed causes 22 random amino acids to be introduced before it encounters a stop codon at position 89, truncating the protein by 33 amino acids (27%). However, as we observed a functional induction, it is unclear if this data is correct and the frameshift is actually present.
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