Difference between revisions of "Team:Wageningen UR/Demonstrate"

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                                <b>Figure 5:</b> CpxR dimerization visualized using eYFP (left) and mVenus (right), with a L-arabinose concentration of 0.2% w/v and different activator (KCl) concentrations over time.
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A proven-to-work affibody against rabbit IgG [4] was fused to the N-terminus of Cpx regulon CpxP (which resides in the periplasm). To determine if IgG can induce the Cpx pathway <mark><a href="http://parts.igem.org/Part:BBa_K2387017">(BBa_K2387017)</a></mark>, the outer membrane of <i>E. coli</i> has to be removed, as IgG cannot penetrate the outer membrane. This is done through a method called spheroplasting. To prevent CpxP from freely titrating away from the membrane it is tether to the inner membrane by fusion to a transmembrane maltose-binding protein (MBP) mutant [5]. This fusion was placed under control of the IPTG inducible <a href="http://parts.igem.org/Part:BBa_K864400">tac promoter(BBa_K864400)</a>
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A proven-to-work affibody against rabbit IgG [4] was fused to the N-terminus of Cpx regulon CpxP (which resides in the periplasm). To determine if IgG can induce the Cpx pathway <a href="http://parts.igem.org/Part:BBa_K2387025">(BBa_K2387025)</a>, the outer membrane of <i>E. coli</i> has to be removed, as IgG cannot penetrate the outer membrane. This is done through a method called spheroplasting. To prevent CpxP from freely titrating away from the membrane it is tether to the inner membrane by fusion to a transmembrane maltose-binding protein (MBP) mutant [5]. This fusion was placed under control of the IPTG inducible <a href="http://parts.igem.org/Part:BBa_K864400">tac promoter(BBa_K864400)</a>
 
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To rapidly translate the generated signal upon IgG binding, Cpx pathway activation is visualized using BiFC. Split eYFP is used as a reporter, and its N- and C-termini are fused to the C-terminus of response regulator CpxR <a href="http://parts.igem.org/Part:BBa_K2387032">(BBa_K2387032)</a>. These fusions were placed under control of the L-arabinose <a href="http://parts.igem.org/Part:BBa_I0500"> inducible pBAD/araC promoter (BBa_BI0500)</a>.
 
To rapidly translate the generated signal upon IgG binding, Cpx pathway activation is visualized using BiFC. Split eYFP is used as a reporter, and its N- and C-termini are fused to the C-terminus of response regulator CpxR <a href="http://parts.igem.org/Part:BBa_K2387032">(BBa_K2387032)</a>. These fusions were placed under control of the L-arabinose <a href="http://parts.igem.org/Part:BBa_I0500"> inducible pBAD/araC promoter (BBa_BI0500)</a>.
 
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To combine these systems (Figure 6), <i>E. coli</i> K12&#916;CpxP was cotransformed with these systems. <a href="http://parts.igem.org/Part:BBa_K2387017">BBa_K2387017</a> was placed in medium copy number plasmid <a href="http://parts.igem.org/Part:pSB3T5"> pSB3T5</a> to enable this.  
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To combine these systems (Figure 6), <i>E. coli</i> K12&#916;CpxP was cotransformed with these systems. <a href="http://parts.igem.org/Part:BBa_K2387025">BBa_K2387025</a> was placed in medium copy number plasmid <a href="http://parts.igem.org/Part:pSB3T5"> pSB3T5</a> to enable this.  
 
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Cells were grown following the <a href="https://static.igem.org/mediawiki/2017/d/d4/Cpx_Dimerization.pdf">spheroplasting protocol</a>. Cells were induced with 0.2% L-arabinose and 0.05 - 0.2 mM IPTG before growing them at 37 &#8451;C ; cells were activated with 0.1 mg IgG at timepoint 0 min. As a control, non-spheroplasted cells were used; we assume IgG cannot penetrate the outer membrane.  
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Cells were grown following the <a href="https://static.igem.org/mediawiki/2017/d/d4/Cpx_Dimerization.pdf">spheroplasting protocol</a>. Cells were induced with 0.2% L-arabinose and 0.05 - 0.2 mM IPTG before growing them at 37 &#8451;C ; cells were activated with 0.1 mg IgG at timepoint 0 min. As a control, non-spheroplasted cells were used; we assume IgG cannot penetrate the outer membrane. Fluorescence was measured at 27 &#8451;C, spheroplasts are expected to remain for 5 generation before fully regenerating their outer membrane. 
 
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                                 <b>Figure 7:</b> Fluorescence/OD<sub>600</sub> values measured over time. Native <i>E. coli</i> K12&#916;CpxP (JW5558(-)) were used as negative controls for both spheroplasted (Sp.) and non-spheroplasted (non-Sp.) cultures.
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<i>E. coli</i> K12&#916;CpxP containing CpxP-Affibody-MBP fusion <a href="http://parts.igem.org/Part:BBa_K2387025">BBa_K2387025</a> and CpxR-eYFPn-CpxR-eYFPc fusions <a href="http://parts.igem.org/Part:BBa_K2387032">(BBa_K2387032)</a>(CpxR-Ab) were induced with 0.2% L-arabinose (L-ara) and 0.05 - 0.2 mM IPTG. 0.1 mg IgG was added at time-point 0. Spheroplasted and non-spheroplasted cells were induced and activated equally. 
 
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Revision as of 10:37, 1 November 2017

Demonstrate

Here we show how we brought individual lab projects together and how we implement them in our device! We performed experiments in which we show that our cells are still viable after drying (which means that they can safely be shipped and still work properly), and that we can measure fluorescence in blood serum!

We combined the "Specific Visualization" and "Fluorescent Protein" modules, by which we aimed to optimize the fluorescent signaling system of Mantis. Furthermore we combine the "Signal Transduction" and "Specific Visualization" modules to directly measure antigens by coupling the affinity molecule, Cpx signal transduction and Bimolecular Fluorescence Complementation (BiFC) specific visualization.


Cell Drying

In order to ship our bacterial system to the local healthcare centre (and to be used in the field later) we need to dry the cells in order to be able to ship them safely and keep them viable.

The first experiment consisted of making an overnight culture of E. coli DH5α. Different amounts of Kaolin clay were added to 2 mL of this culture (in duplo) and left to dry overnight in a flow cabinet on a petri-dish. The next day the cell/clay mixture was scraped off the petridish and resuspended in 1 ml of SOC medium. Of this suspension, 100 µl was put on LB agar plates and left to incubate overnight at 37ºC. The next day, the amount of colonies were counted and compared to the control, to which no Kaolin clay was added. This experiment was repeated under identical conditions, but the dry cell/clay mixture was resuspended and put on plates after 2 weeks of waiting.

In Figure 1, the amount of colonies normalised with regards to the control sample are given on the Y-axis. A clear difference in the survival of the bacteria that were revived after 1 day when compared to the rest can be observed. The sample to which 0.1 grams of clay was added to the cell culture has a significant higher survival rate compared to the control sample. This result gives an indication that drying cells in a Kaolin clay matrix is an improvement over drying cells without a clay matrix.

However, when samples were kept dry for 2 weeks at room temperature there seems to be no significant difference in the survival rate compared to the control sample.

Figure 1: Relative survival rates of E. coli dried in a clay matrix as opposed drying without cell matrix.

Cell Viability and Fluorescence in Blood Serum

Next up, we need to know if our cells can survive and function properly when we add a blood sample to measure antigens. We took several steps to investigate this. First, we analyzed if Escherichia coli could grow in (dilutions of) horse blood serum. To do this, we grew cultures with several ratios of Lysogeny Broth (LB) to horse blood serum overnight. The results can be found in Figure 2.

Figure 2: E. coli is grown in LB with added horse blood serum in different ratios. Cells were grown overnight in 2 mL cultures and the optical density at 600 nm was measured after 18 hours.

We show that E. coli is able to grow in horse blood serum concentrations up to 75%! This means that our cells would be viable when a small amount of growth medium is added before measuring, which prevents a big dilution of the antigen and, subsequently, a lower fluorescent signal.

In addition, we tested if fluorescence can be measured in blood. We grew E. coli K12 containing eYFP under control of the araC/pBAD promoter overnight in LB. These cells were centrifuged and resuspended in 1 mL of LB with added horse blood serum (the same dilutions were used as in the viability test above) and YFP was matured at 30 °C. Fluorescence was measured after six hours.

Figure 3: eYFP fluorescence measured in E. coli resuspended in several LB to horse blood serum ratios. eYFP was excited at 512 nm and measured at 528 nm. Measurements taken after 6 hours.

Here we show that fluorescence can be measured in all blood serum dilutions, whereas the negative controls containing no eYFP show negligible fluorescence!


Improving the Fluorescent Signal

We visualize antigen binding using the Cpx pathway by fusing split fluorophores to interacting proteins. Through a combination of wet- and dry-lab work, we found that a system based on CpxR dimerization yields the best results using bimolecular fluorescence complementation (BiFC) (Figure 4). We used eYFP, split after amino acid 154, as the reporter. This is a commonly used fluorescent reporter in BiFC [2].

We aim to improve this reporter, both in signal intensity and response time. During our "Fluorescent Protein" project we tested a number of fluorescent proteins, of which mVenus showed the shortest maturation time. Furthermore mVenus is designed to have a fast and efficient maturation time [3], exactly what we need!

Also, our Cpx pathway model showed that several interactions of the Cpx pathway visualization can be improved, of which using a fluorescent protein with a decreased maturation time was the most feasible to attempt in a laboratory setting.

We fused mVenus-termini to the C-terminus of CpxR in the same fashion as we did with eYFP and transformed this to E. coli K12. Experiments with mVenus were performed using the same protocol with optimal induction and activation parameters used during experiments with eYFP, and can be found here.

Figure 5: CpxR dimerization visualized using eYFP (left) and mVenus (right), with a L-arabinose concentration of 0.2% w/v and different activator (KCl) concentrations over time.

The results show that usage of mVenus over eYFP as a reporter protein increases the produced fluorescent signal some five times! Unfortunately, the background signal also increases a lot, which means we lose specificity of our response. We hypothesize that the maturation rate of mVenus is too high, which means that many non-specific interactions become irreversible, leading to high fluorescent signals even when no activator is present. This means that mVenus is not a suitable candidate to visualize antigen binding within our diagnostic.

During this project, more reporter proteins were tested. Unfortunately we didn’t have time to test these in the CpxR dimerization setup. At this moment, we recommend testing sfGFP as a reporter for antigen binding. We found that sfGFP is thermostable, i.e. it matures efficiently at high temperatures, while still being one of the fastest and brightest reporters we tested. You can check these experiments here.


Directly Visualizing Antigen Binding

Here we test a direct coupling of the projects “Signal Transduction” and “Specific Visualization”, where we express the whole detection system in one cell, from affinity body to BiFC (Figure 6). While we induced Cpx activation by addition of KCl (to mimic antigen presence), here we actually measure antigen binding.

A proven-to-work affibody against rabbit IgG [4] was fused to the N-terminus of Cpx regulon CpxP (which resides in the periplasm). To determine if IgG can induce the Cpx pathway (BBa_K2387025), the outer membrane of E. coli has to be removed, as IgG cannot penetrate the outer membrane. This is done through a method called spheroplasting. To prevent CpxP from freely titrating away from the membrane it is tether to the inner membrane by fusion to a transmembrane maltose-binding protein (MBP) mutant [5]. This fusion was placed under control of the IPTG inducible tac promoter(BBa_K864400)

To rapidly translate the generated signal upon IgG binding, Cpx pathway activation is visualized using BiFC. Split eYFP is used as a reporter, and its N- and C-termini are fused to the C-terminus of response regulator CpxR (BBa_K2387032). These fusions were placed under control of the L-arabinose inducible pBAD/araC promoter (BBa_BI0500).

To combine these systems (Figure 6), E. coli K12ΔCpxP was cotransformed with these systems. BBa_K2387025 was placed in medium copy number plasmid pSB3T5 to enable this.

Cells were grown following the spheroplasting protocol. Cells were induced with 0.2% L-arabinose and 0.05 - 0.2 mM IPTG before growing them at 37 ℃C ; cells were activated with 0.1 mg IgG at timepoint 0 min. As a control, non-spheroplasted cells were used; we assume IgG cannot penetrate the outer membrane. Fluorescence was measured at 27 ℃C, spheroplasts are expected to remain for 5 generation before fully regenerating their outer membrane.

Figure 7: Fluorescence/OD600 values measured over time. Native E. coli K12ΔCpxP (JW5558(-)) were used as negative controls for both spheroplasted (Sp.) and non-spheroplasted (non-Sp.) cultures. E. coli K12ΔCpxP containing CpxP-Affibody-MBP fusion BBa_K2387025 and CpxR-eYFPn-CpxR-eYFPc fusions (BBa_K2387032)(CpxR-Ab) were induced with 0.2% L-arabinose (L-ara) and 0.05 - 0.2 mM IPTG. 0.1 mg IgG was added at time-point 0. Spheroplasted and non-spheroplasted cells were induced and activated equally.

References

  1. Zohar-Perez, C., Chernin, L., Chet, I., & Nussinovitch, A. (2003). Structure of Dried Cellular Alginate Matrix Containing Fillers Provides Extra Protection for Microorganisms against UVC Radiation Structure of Dried Cellular Alginate Matrix Containing Fillers Provides Extra Protection for Microorganisms against UVC Radiation. Radiation Research Society, 160(2), 198–204.
  2. T. Kerppola, “Bimolecular fluorescence complementation (BiFC) analysis as a probe of protein interactions in living cells,” Annu. Rev. Biophys., vol. 37, pp. 465–87, 2008.
  3. Nagai, T., Ibata, K., Park, E. S., Kubota, M., & Mikoshiba, K. (2001). A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nature Biotechnology, 20, 1585–1588.
  4. Nilsson, Björn, et al. "A synthetic IgG-binding domain based on staphylococcal protein A." Protein Engineering, Design and Selection 1.2 (1987): 107-113.
  5. Raivio, Tracy L., et al. "Tethering of CpxP to the inner membrane prevents spheroplast induction of the Cpx envelope stress response." Molecular microbiology 37.5 (2000): 1186-1197.
  6. Fikes, John D., and P. J. Bassford. "Export of unprocessed precursor maltose-binding protein to the periplasm of Escherichia coli cells." Journal of bacteriology 169.6 (1987): 2352-2359.