Chromate Biosensor

Summary

Introduction

Background

Stainless steel is an alloy of different metals and apart from the base material, iron, contains a large proportion >11% of chromium. There is a wide range of stainless steels for a variety of different applications, ranging from surgical steel to alloys used in the aeronautic industry. For our application we assumed stainless steels that are used in bulk production of standard kitchen knives with a chromium content between 12% and 14%. We also utilise the fact that chromate is a salt containing chromium. Bone incisions and kerfmarks, distinctive marks left behind, are current indicators for different types of weapons such as saws (1) or knifes.

We will use pig bones to investigate incisions by stainless steel.

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Initial Ideas

At the beginning of the project we met with Dr. Lucina Hackman, an expert in dismemberment cases at the University of Dundee. In dismemberment cases it is obvious enough that a body has been dismembered. However, if the body is not found for a long time and the only remains are bones, it is hard to tell if a person has been stabbed or if an animal was chewing on the bone. This is where a chromate biosensor would be useful. By detecting minute amounts of chromate on a bone, the use of a weapon may be inferred which will justify searching for trace evidence.

We found that a chromate detector had already been submitted by the Beijing Institute of Technology 2013 iGEM team, where they had created a biobrick for this (BBa_K1058008), shown in Figure 1. The biobrick is a modified version of a chromate resistance operon of ochrobactrum tritici 5bvl1(2,3).

The science behind Chromate resistance: Ochrobactrum tritici 5bvl1 was discovered in 2004 in a chromate contaminated wastewater treatment facility. A specific characteristic of this strain was that it could both, reduce Cr(VI) and be resistant to it at the same time. Previously characterised strains were either resistant or could reduce (2). Over the last 10 years the group that discovered it started working out the underlying mechanisms of this set of characteristics. A transposon located set of genes that lead to chromate resistance (TnOtChr) was uncovered. This operon, expressed from the pChr promoter led to the expression of the genes ChrA, ChrB, ChrC, and ChrF. ChrA was found to express and efflux pump, whereas ChrB was identified as being a chromate-inducible regulator of pChr. Additionally, ChrC and ChrF were found to confer tolerance to superoxides. Deleting ChrC and ChrF was shown to have no significant effect on chromate resistance which at first seemed unusual, but considering that Cr(VI) is often reduced via Cr(V) and Cr(IV) intermediates which can lead to the generation of superoxides, these 2 genes are suspected to act as a second line of defense (4).

Figure 1: Plasmid map of the original biobrick system, (BBa_K1058008).

We aimed to modify and improve this existing biobrick by taking the brick apart and use its constituent parts in a new arrangement, Figure 2. This should have the advantage that each part can be modified individually and the level of GFP and/or the repressor can be modulated more easily by simply cloning it into a higher or lower copy number plasmid.

Figure 2: Plasmid map of the modified biobrick system, **(BBa_K1590003**, **BBa_K1590004)**.

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What did we do?

To ensure that the modified system was valid we used mathematical modelling to compare the original biobrick and our modified system, Figure 3. We also looked at GFP production with varying levels of chromate added to the system, Figure 4. The models of both the original and modified pathways revealed two key points; the modified pathway is just as reliable as the original pathway and the level of GFP produced will not be changed. However the response will be faster for increasing levels of chromate added to the system.

Figure 3: Comparing the likelihood of false positives, in terms of leakiness, of both systems in the case of GFP output. The figure shows that both systems are as reliable as each other.

Figure 4: GFP production with varying levels of chromate present showing that the more GFP the faster the response.

Learn more about modelling ↑

The models allowed us to ensure that it will be valid to use our modified system. In the lab to test the system we used the two compounds potassium chromate and potassium dichromate, as well as the two controls, Chromium(III)Chloride and the isostructural potassium sulphate and observed if it had any influence on the level of fluorescence. We tested concentrations between 10nM and 100µM as recommended by literature (5). Our cloning strategy focused on taking this brick apart and use its constituent parts in a new arrangement.

The newly arranged system was then investigated by firstly comparing the GFP output for different concentrations of chromate, Figure 5. Cell density over time was also considered using chromate and other similar substances in response to the system, Figure 6. From these experiments it was seen that at a specific time both fluorescence and cell density increased, when the cell density reached exponential growth. this inferred that fluorescence only increased once the cell density increased, further experiments would have to be done to try and disconnect these two variables. After these experiments we then wanted to compare the original biobrick and the modified two plasmid system.

Figure 5: Plot showing change in fluorescence over time of results of plate reader experiment after exposing MG1655 + BBa_K1058008 to a range of concentrations between 10 nM and 100 µM of K₂CrO₄, K₂Cr₂O₇, CrCl₃, and K₂SO₄. One measurement was made every 10min across 16h.

Figure 6: Plot showing change in cell density over time of results of plate reader experiment after exposing MG1655 + BBa_K1058008 to a range of concentrations between 10 nM and 100 µM of K₂CrO₄, K₂Cr₂O₇, CrCl₃, and K₂SO₄. One measurement was made every 10min across 16h.

The original biobrick and our modified two plasmid system were compared by looking at the differences in GFP output. Different concentrations of the biobrick and the modified system were loaded onto a gel and blotted against GFP, Figure 7 and Figure 8. It was found that GFP was produced in the absence of chromate for both systems. This is perhaps due to an error in the original biobrick. This result was unexpected and further investigation into why this occurred is required.

Figure 7: Top: anti-GFP: Single colonies of JM110 + pSB1C3-Pchr-gfp (A) and MC1061 + pSB1C3-Pchr-gfp (B) were prepared and diluted to a suitable concentration for western blotting against GFP. pSB1C3 was included as a negative control, and *hydA - 6-His* as a positive control. Bottom: anti-6-His - ChrB: JM110 pUniprom-*chrB* and JM110 pUniprom-*chrB* (opt) were prepared accordingly. pSB1C3 was included as a negative control, and *hydA - 6-His* as a positive control.

Figure 8: Western blot against GFP: Single colonies of MG1655, containing all 4 combinations, pUniprom-*chrB* - pSB1C3-Pchr-gfp (A), pUniprom-*chrB* - pSB1C3-Pchr-gfp (B), pUniprom-*chrB* (opt) - pSB1C3-Pchr-gfp (A), and pUniprom-*chrB* (opt) - pSB1C3-Pchr-gfp (B) were used to inoculate 5ml of LB broth supplemented with 100 ^µg/_ml chloramphenicol and 100 ^µg/_ml ampicillin. After 16h of incubation at 37°C with agitation at 200rpm, each sample was subcultured into 5ml of fresh, equally supplemented LB and cells were grown for 2 hours more. 1ml of the subculture was then retrieved and pelleted. The pellet was resuspended in 1ml PBS. 100 µl of these samples was mixed with 100 µl laemmli buffer, and the sample boiled for 10 min. The indicated volume of each sample was loaded on a SDS gel (12% acrylamide). pSB1C3, and pUniprom were included as a negative control, and PmanA-gfp as a positive control. The blot against 6-His – ChrB was without success and is not shown. No chromate of any kind was added to the medium previous to this blot, hence no expression of GFP was expected.

Learn more about lab work ↑

To complement our chromate biosensor, we originally aimed to find out how much chromate would be left on an incision in a bone from a stab of a certain force. However this was quickly turned around to measure the wear of bone instead of stainless steel. By calculating the force required to make the incision, this could potentially indicate the size or strength of a possible suspect, while the sliding distance would correspond to the length of the blade. The bone incision experiments allowed us to create a relationship between the applied force, sliding distance and wear volume when a stainless steel knife collides with bone. The results showed that the volume will increase as the length of the blade or the force increases, Figure 9. However some irregularities were noticed and could be due to experimental error with the quitment shown in Figure 10. You can see Hannah demonstrating the method used in our STV appearance.

Figure 9: A graph showing the relationship between the volume of a bone incision, length of the blade and force of stabbing.

Figure 10: The Intron 4204 with the knife attached and bone placed on stand below.

Learn more about bone experiments ↑

What next?

Since most of the parameters were assumed in the mathematical models then there is room for error. Practical experiments could be done to produce values for the kinetic parameters or sensitivity analysis done to compare varying kinetic parameter values. Probing the concentration range for detecting different chromate salts will be essential to feed back into the modelling which is currently largely based on assumptions. This would ensure that the models are more realistic and therefore give a more valid description of the systems.

Figure 11: Further work would include testing and validation of our methods.

The next step for this project is to find an ideal concentration of ChrB in relation to pChrGFP to work in a balance way of expression/repression. This involves finding plasmids with an appropriate copy number and promoters of appropriate strength. The modularity of this system makes this process easier. Once a concentration range has been established, we can test the system by using actual shavings of stainless steel. A problem that might occur is that the chromium contained in the alloy is in the wrong oxidation state. Oxidising chromium to yield Cr(VI) can be done by subjecting it to a strong acid. The solution will have to be neutralised again to be used as an additive for an experiment. The question is then if the concentration is still high enough to induce expression of GFP in our system. Good insights into the amount of steel retained after sharp-force trauma and blunt-force trauma on bones can be found in a series of papers (6,7). Of great importance will also be potential contaminants. Hence testing the system with similar compounds will be one test. Checking for the prevalence of inducing compounds in or around bones is essential to see how prone the test is to leading to false negatives.

To remove doubts in the data from the bone incision experiments, due to possible experimental error we should repeat the experiments. This would ensure that the experiments are reproducible and reliable.

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References

Saville PA, Hainsworth SV, Rutty GN. Cutting crime: the analysis of the "uniqueness" of saw marks on bone. Int J Legal Med 2007; 121(5): 349-357.
Branco R, Alpoim MC, Morais PV. Ochrobactrum tritici strain 5bvl1 - characterization of a Cr(VI)-resistant and Cr(VI)-reducing strain. Can J Microbiol 2004; 50(9): 697-703.
Branco R, Morais PV. Identification and Characterization of the Transcriptional Regulator ChrB in the Chromate Resistance Determinant of Ochrobactrum tritici 5bvl1. PLoS ONE 2013; 8: 77987.
Branco R, Chung AP, Johnston T, Gurel V, Morais P, Zhitkovich A. The chromate-inducible chrBACF operon from the transposable element TnOtChr confers resistance to chromium(VI) and superoxide. J Bacteriol 2008; 190(21): 6996-7003.
Branco R, Cristovao A, Morais PV. Highly Sensitive, Highly Specific Whole-Cell Bioreporters for the Detection of Chromate in Environmental Samples. PLoS ONE 2013; 8(1): e54005.
Pechníková M, Porta D, Mazzarelli D, Rizzi A, Drozdová E, Gibelli D, Cattaneo C. Detection of metal residues on bone using SEM-EDS. Part I: Blunt force injury. Forensic Sci Int 2012; 223(1): 87-90.
Gibelli D, Mazzarelli D, Porta D, Rizzi A, Cattaneo C. Detection of metal residues on bone using SEM-EDS--part II: Sharp force injury. Forensic Science International 2012; 223(1): 91-96.

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Team:Dundee/Forensic Toolkit/Chromate Fiona