Difference between revisions of "Team:Harvard BioDesign/Platform"
| Line 222: | Line 222: | ||
</p> | </p> | ||
<br> | <br> | ||
| − | <img src="https://static.igem.org/mediawiki/2015/ | + | <img src="https://static.igem.org/mediawiki/2015/5/51/Harvard_SBOL_Diagram.png" alt="SBOL Diagram" style="width:890px;height:494px;margin-top:-10px;margin-bottom:35px;border:1px solid darkgrey;"/> |
<p> | <p> | ||
The system was arranged on two plasmids so that we could modulate the amount of adhesive fimH component with respect to the other pili structural genes. Additionally, we found that it was easier to customize fimH on a smaller plasmid backbone since it eliminated cloning issues encountered when the entire fim operon was located on a single plasmid. Finally, we selected pAra (LINK HERE) because it is a titratable promoter that allows for controlled expression of pili structural genes over a broad range of inducer concentrations (reference). Rhamnose and arabinose were added into broth cultures of E.coli containing both of the plasmids to create synthetic type I pili. | The system was arranged on two plasmids so that we could modulate the amount of adhesive fimH component with respect to the other pili structural genes. Additionally, we found that it was easier to customize fimH on a smaller plasmid backbone since it eliminated cloning issues encountered when the entire fim operon was located on a single plasmid. Finally, we selected pAra (LINK HERE) because it is a titratable promoter that allows for controlled expression of pili structural genes over a broad range of inducer concentrations (reference). Rhamnose and arabinose were added into broth cultures of E.coli containing both of the plasmids to create synthetic type I pili. | ||
Revision as of 11:12, 18 September 2015
BactoGrip: Our Platform
This summer we dedicated ourselves to building a modular, durable, biomimetic platform for synthetic biologists who need to control bacterial adhesion. We developed a set of assays to reliably determine how our system is functioning and selected relevant positive and negative control strains to differentiate real from imaginary results. Using the tools of synthetic biology, we created BioBricks which can be integrated into complex biological circuits where specific interaction with the physical environment is a must. Furthermore, we created a workflow that can be used to integrate any desired binding specificity into our system, and modified the native protein so that it is easy to work with and measure under diverse experimental conditions. We call our system BactoGrip, and we hope you will find it as gripping as we do.
Strains and Assays
Before we could begin engineering the Fim system (link to Ben's description of the operon) we needed to establish a positive and negative baseline for the experiments we'd use to categorize our constructs. We selected two strains from the Yale's Keio collection (link) of nonessential gene knockouts which are each missing one of the recombinases that comprises the fim “switch” (link to Ben's background). We hypothesized based on the literature that a knockout for the fimB recombinase (Keio strain name JW4276) will not produce pili because fimE is biased to switch in the direction of “on” to “off” (switchAndInteractionfimEandB). When fimB is absent, fimE would switch the operon “off”. Contrarily, a knockout for fim E (Keio name- JW4276) will overproduce pili because fimB switches both “on” to “off” and “off” to “on”, so the operon will be “on” more than it would if both were present.
To confirm this hypothesis we used an agglutination assay (protocol here link to protocol) from the literature which is standard for measuring the expression of Type 1 Pili (Klemm, Schembri, Stentebjerg-Olesen, Hasman, Hasty 1998).
This protocol tests whether a culture of E. coli cells can agglutinate--clump together--a substrate which contains the binding partner (link to background) of fimH, mannose sugar. We chose to try to clump together S. cervisiae (baker's yeast) because it expresses mannose on its cell surface, is easy to grow in lab, and has been used in this assay before (Eshdat, Speth, Jann 1981).
We found that the fimE KO we hypothesized would overproduce type 1 pili indeed agglutinated the yeast, as indicated by a white clump at the bottom of the tube. The fim B KO samples showed no sign of clumping and were completely opaque:
To confirm these results, we examined equal volumes of OD standardized (protocol link) agglutinated samples from either strain under light microscopy. Slides were prepared according to the slide preparation protocol (link) and to visualize the bacteria and yeast, we stained the slides according to the Gram Stain protocol (link).
These photos are representative of the morphology of each sample. The large purple circular cells are S. cerevisiae and the small pink cocci are E. coli:
At this point we were confident in the agglutination assay’s ability to detect the presence of pili via mannose-mediated adhesion. However, we hypothesized that our future modifications of the fimH adhesin might lead to weakened mannose binding strength even while the pili were still properly assembling. We required a diagnostic assay that would detect whether type 1 Pili were present on the cell surface even when fimH no longer bound to mannose. So we conducted extensive literature review and developed the (Pili Purification Protocol link) with the aim of a rapid test to provide a “yes” or “no” answer to this question.
We first tested our assay on our control strains, growing both the overproducer and null strain overnight in LB liquid culture, then OD standardizing (protocol) to ensure our results were not dependent on variable cell growth. Then a volume of each sample was spun down at low speed (so as not to lyse the cells), washed once in DPBS to remove secreted proteins, and resuspended in a smaller volume of DPBS to concentrate the signal in later steps. These samples were then heat treated for 20 minutes at 60°C to shear-off the pili from the cell surface and spun down again at low-speed so the cells would pellet but the sheared pili would remain in solution. This pili-containing supernatant was then denatured using formic acid, boiled in 1x Lamelli’s buffer and run according to the SDS-PAGE protocol (link). The resulting gels were stained according to the R-250 Coomassie protocol (link) and compared at the molecular weight of Type 1 Pili’s structural subunit protein, fimA, which is most abundant. We hypothesized that the pili overproducing fimE would show a strong band at this weight compared to the null fimB strain. The resulting gel is below:
Caption: “A band is present at the expected weight in the purified pili of the fimE overproducer. We tried testing the purification with and without formic acid acid denaturation. The fimA band is stronger in the formic acid treated sample which indicates that the pili are insoluble and need to be broken up for detection”
Indeed, we see a dark band at 16.5 kD, the molecular weight of fimA, but only for the fimE overproducing strain, not for our negative control. This shows our pili purification method gives a clear signal to detect pili production, and can be used to characterize our modified Type 1 Pili and by future teams using our system. With functional assays and positive and negative controls, we felt confident that we had the tools necessary to begin engineering control over pili expression.
Construct Design
Our type I pili-expression system is distributed across two plasmids wherein the pili genes on each are expressed under different, inducible promoters. The fimH gene was amplified from the E. coli K12 genome and placed under the control of a rhamnose-inducible (Bba_K902065) promoter with a strong ribosome binding site (B0034). The remaining type I pili structural and transport genes from the fim operon were amplified from E. coli K12 and placed under an arabinose inducible (BBa_I13453) promoter with the native ribosomal binding sites. Gibson assembly was used to place the parts into pSB1C3, illegal cut sites were eliminated to make the parts RFC10 compatible, and each circuit was transferred into low copy number expression plasmid (LINKHERE to uneditable Benchling sequences).
The system was arranged on two plasmids so that we could modulate the amount of adhesive fimH component with respect to the other pili structural genes. Additionally, we found that it was easier to customize fimH on a smaller plasmid backbone since it eliminated cloning issues encountered when the entire fim operon was located on a single plasmid. Finally, we selected pAra (LINK HERE) because it is a titratable promoter that allows for controlled expression of pili structural genes over a broad range of inducer concentrations (reference). Rhamnose and arabinose were added into broth cultures of E.coli containing both of the plasmids to create synthetic type I pili.
We introduced protein fusions to three discrete sites of within the FimH adhesin protein. These included the N-terminus immediately after the secretory peptide as well as after amino acids 225 and 258 from this site.
These selections were based on previous literature wherein small peptide fusions were introduced successfully at these sites. Sites 225 and 258 showed strong evidence in the literature that small fusions such as his-tags were expressed and functional on assembled pili.Conversely, prior work at the N-terminus has not shown that peptide fusions could be expressed and properly assembled at this site., Thus, we added novel peptide fusions to these sites in fimH using site-directed mutagenesis to customize the adhesive properties of type I pili. Additionally, we sought to determine whether these sites could be modified to make fimH generalizable to myriad localization and binding properties in the iGEM community.
Validating Expression
We established that our engineered system could control pili expression by performing an agglutination. Following the protocol, we mixed cultures of our induced and uninduced plasmid-containing fimB knockouts with S. cervisiae yeast along with controls. We found that our plasmids recovered agglutination in the negative control strain and that this agglutination was dependent on the addition of inducer molecules. This shows both that we have control over expression of the biosynthetic machinery and the fimH adhesin, and that the resulting pili were functional as determined by a standard assay for mannose binding. This assay was performed in biological triplicates with the same result. Image below:
Making it Measureable
For our controlled adhesion system to be a useful platform for other iGEM teams and synthetic biologists, we needed to develop a method to reliably measure expression of our recombinant adhesin, fimH. To that end we inserted a His Tag into the 225 fusion site via site-directed mutagenesis (protocol link) and tested whether we could detect induction via an α-His Western Blot (link to protocol). Cultures of our His Tagged-fimH plasmid containing pili knockout bacteria were OD standardized (protocol) and separated into two subcultures, one of which was induced with Rhamnose, the other uninduced. Samples taken at three timepoints show increasing expression of fimH in the induced culture but not the uninduced. This demonstrates that we can express recombinant fimH and that the His Tag gives us traction to measure expression.
In principle, the His Tag on fimH could also be used to quantify recombinant expression in assays like whole-cell ELISA, both integrated with the pilus shaft and separately. In the future we hope to test the efficacy of these assays for determining quantitative expression levels. Additionally, the His Tag will allow future teams to purify recombinant pili using Nickel NTA purification (link) for use in vitro.