Team:Harvard BioDesign/Metal
Applications: Mining and Materials
In nature, bacteria bind to abiotic surfaces like metal to fulfill a plethora of evolutionary purposes. For the synthetic biologist, harnessing this ability is one of the most promising directions for making biology into a technology that can solve pressing global problems, from heavy metal pollution and sustainable mineral extraction to biomaterials. We applied BactoGrip to metal binding as an in vitro test of our platform, motivated by the potential to use it to extract valuable minerals or heavy metal pollution from water and create a specific biological-inorganic interface for the programmable materials of the future. We choose to try using our pili binding to stainless steel and Nickel to this end.
Construct Design
A nickel binding His Tag and a stainless steel binding Metal Binding Domain (MBD)6 were located in the literature and inserted into fusion sites of fimH via site directed mutagenesis (link to construct design, site-directed mutagenesis protocol). These fimH constructs were cotransformed with the fim operon and expressed in fimB pili knockouts.
Validating Expression
Pili Purifications were performed to validate pili expression of these metal binding constructs. Cultures were grown to a standard OD, induced with either rhamnose, arabinose, both or neither (protocol), and pili-purified (protocol). Samples were run on an SDS-PAGE gel and stained with Coomassie G250 (protocol). Images below:
We saw pili expression, as shown by a fimA band, in both the arabinose induced and doubly induced cultures of both the MBD and His-Tag. We suspect the reason why the arabinose-only induced culture showed this band is either because our rhamnose promoter may have enough basal expression to produce pili, or fimA accumulated in the periplasm may be released into solution by the heat treatment step of pili purification. This is an area of ongoing research in the lab.
Testing Nickel Binding
We set out to determine whether our His Tag constructs would bind to nickel. We used an assay similar in principle to the agglutination assay (nickel agglutination protocol) which would detect whether our bacteria could bind to Nickel-NTA magnetic microbeads and aggregate them or remain in suspension. OD standardized volumes of induced and uninduced cultures were added to an equivalent volume of suspended Nickel-NTA beads, agitated, and allowed to rest.
We found that our His Tag fimH plasmids were able to agglutinate nickel beads, while a Wild Type control was not. This suggests control over adhesion to Nickel-NTA magnetic beads:
The ability to remove nickel particles from a solution could be extremely useful in waste water purification and pollution remediation. This experimental setup is almost identical to how we imagine our nickel binding system could be used in the real world. A sample of our E. coli could be added to a contaminated sample of water, the sample could be agitated, and any heavy-metal contaminants would fall out of solution with the bacterial clump. Future work can elaborate the toolbox of contaminant-binding peptides so that a range of harmful products could be purified. Furthermore, this process might just as easily be applied to mining valuable minerals from water, where binding peptides for rare and precious materials could be added to fimH, the resulting induced cultures mixed with a water sample containing the material of interest, and the bacterial clump extracted as if “mined” out of water. Our biological approach could be superior to existing mining techniques which require toxic chemicals and generate environmentally hazardous waste products. With biological extraction, the only waste would be a completely biodegradable and present in the water anyway (bacteria are present everywhere). These directions are areas of ongoing research in the lab.
Testing Steel Binding: Part 1
Specific binding to stainless steel was assayed in two ways. The first measured the amount of bacteria left bound to stainless steel microspheres after repeated wash steps to remove any bacteria interacting nonspecifically. Overnight cultures of our MBD-fimH with the operon were induced according to our induction protocol (link). Cultures were OD standardized (link) and equalized volumes were added to suspended stainless steel microspheres (manufacturer?). The beads were sedimented by differential centrifugation and washed repeatedly, centrifuging each time to pull down any bound bacteria and rinsing away the nonspecifically bound bacteria in the supernatant. After sufficient washing, the remaining beads were resuspended in Lamelli’s buffer and boiled to break open cells and denature proteins (protocol) . These samples were then run on an SDS-PAGE gel (protocol) and stained with Coomassie G-250 (protocol). We hypothesized that the resulting gel would show heavy bands in the lanes from samples which adhered specifically to the stainless steel beads, because there would be a higher concentration of cells and cellular proteins where the bacteria bound specifically and clung through the wash steps. This assay was performed in biological triplicates and a representative result is depicted below:
If the lane is empty, it indicates little or no bacteria were stuck to the steel beads after the wash process. If the lane has protein bands, it indicates that bacteria adhered to the stainless steel beads strong enough to persist through the wash process and show protein in the whole cell lysate. We see strong bands in only two lanes, the MBD-fimH induced with only arabinose, the operon inducer, and the MBD-fimH induced with both arabinose and rhamnose, which has darker bands suggesting stronger adhesion. The binding we see in the arabinose-only lane is once again indicative of leaky expression of the rhamnose promoter, and works is ongoing to optimize the expression system for increased control. This preliminary result was a promising indication of successful stainless steel adhesion, but we wanted to more evidence to characterize the part.
Testing Steel Binding: Part 2
To obtain verify our above result we developed an assay protocol which would measure adherence of our steel Bactogrip E. coli to a stainless steel substrate. As above, we grew overnight cultures of steel Bactogrip and controls (protocol) induced overnight (protocol) and OD-standardized (protocol). Equalized volumes of sample were “dotted” in triplicate on clean stainless steel coupons, allowed to sit for a short period to interact with the surface, washed three times with equal volumes of TTBS to remove the dots and then washed for an hour in TTBS to disrupt nonspecific binding. Coupons were then blocked with TTBS-Milk and antibody stained as if performing a western blot. We hypothesize that an α-LPS primary stain (source) which illuminates E. coli generally will show stronger luminescence, and thus higher E. coli concentration, on the dots containing our induced MBD-fimH compared to controls. The assay was performed in biological duplicate and a representative result is shown below:
Indeed, we see the induced MBD-fimH bind much stronger than both the uninduced and control strains in all technical replicates. This validates our pull-down assay result and demonstrates that we can control adhesion to stainless steel.
In the future we will pursue more quantitative adhesion assay methods inspired by the superb biofilm / adhesion assay development work achieved by our collaborators, WPI. We see controlled stainless steel adhesion as a useful tool for developing a biological-inorganic interface for composite biomaterials, and hope future iGEM teams can make use of it in ways we could never imagine.