Difference between revisions of "Team:Wageningen UR/Description/Regulation"
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<h2><b>Overhauling population dynamics to improve toxin production</b></h2> | <h2><b>Overhauling population dynamics to improve toxin production</b></h2> | ||
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| − | In our quest to save the honey bees from <i>Varroa destructor</i> we envisioned using <i>Bacillus thuringiensis</i> cry toxins. When activated and concentrated near the surface of cell membranes, Bt-toxins form pores inside the membrane, lysing the cell as a result<sup><a href="#fn1" id="ref1">1</a></sup>. High level constitutive Bt-endotoxin expression in <i>Escherichia coli</i> is known to inhibit growth and possibly kill the producing cells<sup><a href="#fn2" id="ref2">2</a></sup>. | + | In our quest to save the honey bees from <i>Varroa destructor</i> we envisioned using <i>Bacillus thuringiensis</i> cry toxins. When activated and concentrated near the surface of cell membranes, Bt-toxins form pores inside the membrane, lysing the cell as a result<sup><a href="#fn1" id="ref1">1</a></sup>. High level constitutive Bt-endotoxin expression in <i>Escherichia coli</i> is known to inhibit growth and possibly kill the producing cells<sup><a href="#fn2" id="ref2">2</a></sup>. The aim behind the population dynamics subproject is to deliver a system for toxin regulation where production does not impair growth nor population survival of the bacteria. |
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Solutions to the problem of toxic expression are found in mechanisms that regulate protein production to minimize the negative effects on growth and survival<sup><a href="#fn3" id="ref3">3</a></sup>. Inducible expression for instance, is widely used to manually confine toxin expression in time to after the exponential growth phase of bacteria. We propose a regulation system that enables <i>E. coli</i> to separate the growth and the production phase by only expressing recombinant proteins when bacterial cell density is high. <b>We cannot expect beekeepers to measure bacterial growth and induce expression themselves when the time is right</b>. | Solutions to the problem of toxic expression are found in mechanisms that regulate protein production to minimize the negative effects on growth and survival<sup><a href="#fn3" id="ref3">3</a></sup>. Inducible expression for instance, is widely used to manually confine toxin expression in time to after the exponential growth phase of bacteria. We propose a regulation system that enables <i>E. coli</i> to separate the growth and the production phase by only expressing recombinant proteins when bacterial cell density is high. <b>We cannot expect beekeepers to measure bacterial growth and induce expression themselves when the time is right</b>. | ||
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| − | < | + | <h2><b>Quorum Sensing: regulation based on bacterial density</b></h2> |
| − | < | + | For bacteria to regulate toxin production based on the density of bacteria, they need to be able to communicate bacterial density among them. Systems that enable bacteria to do so are called <b>quorum sensing</b> mechanisms. We have adopted one of the best known quorum sensing systems: the lux system originating form <i>Vibrio fischeri</i>. This systems consists of <b><i>luxI</i></b> and <b><i>luxR</i></b> two genes that allow for bacterial density communication. The <b>LUXI</b> protein is a synthase that produces acyl-homoserine lactones (AHLs). AHLs are small compound that diffuse across cell-membranes and function as autoinducers, the molecules that bacteria secrete to signal population density. |
| − | + | A single cell produces insufficient autoinducer molecules to start quorum sensing. When there is a high density of bacteria, all producing AHL, the AHL concentration in the growth medium increases. AHL can then reach high enough cytoplasmic concentrations to effectively bind the LUXR protein. <b>LUXR</b> is a cytoplasmic receptor protein binding AHLs, regulating gene expression depending on whether AHL molecules are bound. | |
| − | A single cell produces insufficient autoinducer molecules to start quorum sensing. When there is a high density of bacteria, all producing AHL, the AHL concentration in the growth medium increases. AHL can then reach high enough cytoplasmic concentrations to effectively bind the | + | <br><br> |
| − | + | To test density dependent expression, a two plasmid quorum sensing system was assembled in and transformed to <i>E. coli</i> DH5alpha cells. One plasmid contains the actual quorum sensing system <a href="http://parts.igem.org/Part:BBa_K546000">BBa_K546000</a> in the <a href="http://parts.igem.org/Part:pSB4K5">pSB4K5</a> backbone, while the other plasmid contains a newly made GFP quorum sensing reporter <a href="http://parts.igem.org/Part:BBa_K1913014">BBa_K1913014</a> in the <a href="http://parts.igem.org/Part:pSB1C3">pSB1C3</a> backbone. Bacteria that contain both plasmids should be able to communicate their cell density and produce GFP once cell density gets high enough. The bacteria were indeed found to color green in overnight liquid cultures, preliminary indicating functional quorum sensing and reporting hereof (figure 3). The cells that contain only the reporter plasmid do not give a green pellet, indicating that they do not substantially activate expression of GFP. | |
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| − | + | Figure 3. Cell pellets of 10ml LB cultures of (left) DH5alpha cells containing both the quorum sensing system and the reporter plasmid. (right) DH5alpha cells containing only the reporter plasmid. | |
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| − | Figure 3. Cell pellets of 10ml LB cultures of (left) DH5alpha cells containing both the quorum sensing system and the reporter plasmid. (right) DH5alpha cells containing only the reporter plasmid | + | |
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To follow the dynamics of the quorum sensing, plate reader measurements were done for the previously mentioned 2 plasmid quorum sensing <i>E. coli</i>. Fluorescence intensity was assessed by exciting at 480 nm and measuring light intensity of 510 nm. In addition, the optical density (absorbance at 600nm) was measured to relate fluorescence intensity to cell density (figure 4). | To follow the dynamics of the quorum sensing, plate reader measurements were done for the previously mentioned 2 plasmid quorum sensing <i>E. coli</i>. Fluorescence intensity was assessed by exciting at 480 nm and measuring light intensity of 510 nm. In addition, the optical density (absorbance at 600nm) was measured to relate fluorescence intensity to cell density (figure 4). | ||
| − | Fluorescence over OD<sub>600</sub> is plotted to correct the fluorescence intensity for the amount of cells that produce the fluorescence. Starting from an OD<sub>600</sub> of 0.8 there is a sharp increase in fluorescence intensity per cell for the quorum sensing system. | + | Fluorescence over OD<sub>600</sub> is plotted to correct the fluorescence intensity for the amount of cells that produce the fluorescence. Starting from an OD<sub>600</sub> of 0.8 there is a sharp increase in fluorescence intensity per cell for the quorum sensing system. A very small increase in fluorescence/OD<sub>600</sub> is observed for the reporter only control cells as well. |
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| + | <h2><b>Subpopulation formation</b></h2> | ||
| + | Of course, population wide synchronized toxin overexpression is likely to kill all <i>E. coli</i> cells in the first wave of toxin expression. It would be more beneficial to increase the survival chances of <b>some</b> bacteria by keeping them as non-producers. Cells surviving the production phase would be able to initiate a new growth phase after death of the producing cells. The critical requirement for this is that while most cells display a certain behaviour (toxin production), some cells in the same population behave differently from the rest. A collection of cells that acts differently from the rest of the population is called a <b>subpopulation</b>. | ||
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| + | We hypothesized that a balance between two antagonistic genes can function as the basis for a subpopulation inducing circuit. The working of the dominantly expressed gene determines cell behaviour. | ||
| + | We based our system on <a href="http://parts.igem.org/Part:BBa_I12006">a modified lambda Prm promoter</a>. This promoter is regulated by two cI ‘repressor’ proteins that have opposite effects on transcription of downstream genes. Despite its name, the cI repressor protein from phage λ induces the promoter, whereas the cI repressor protein from phage 434 represses the promoter. These two phage derived genes thus work antagonistically and function as the basis of the subpopulation system reported here. | ||
| + | To actually create a subpopulation it is essential that the levels of the two cI proteins (or at least the ratios between them) can differ from cell to cell. Inspired by persister cell formation, we investigated expressing the two cI genes in one operon behind the same promoter. Difference in the turnover of the two proteins assures that changes in promoter strength induce temporary changes in the ratio between the level of the proteins. | ||
| + | Imagine a system where both cI genes are transcribed at intermediate levels. 434 cI has a much stronger RBS than the λ cI gene and therefore is translated at a higher rate. This leads to a situation where there are more 434 cI than λ cI proteins: 434 cI is dominant. | ||
| + | When the promoter regulating both genes is repressed, both genes will in time reach a new (lower) balance of protein levels. However, when 434 cI is degraded faster than λ cI, 434 cI protein levels will decrease more rapidly. Given the right tuning, this allows λ cI to briefly dominate the system. We hypothesize that small differences (cell age, metabolism etc.) between cells can determine whether λ cI indeed gets the chance to dominate the system. | ||
| + | This principle of protein balance chances through changes in promoter strength is similar to how some cells become persister cells, while other continue growing<sup><a href="#fn5" id="ref5">5</a></sup>. <b>We decided against an attempt to create actual persisters out of safety reasons.</b> Persisters cells are a well studied yet poorly understood example of microbial subpopulations<sup><a href="#fn6" id="ref6">6</a></sup>. Persister cells make up around 1% of the population in the stationary phase<sup><a href="#fn7" id="ref7">7</a></sup>. In the main model for persister cells, persisters differ from other cells in the balance between toxin and antitoxin. In persister cells, the toxin in a toxin/antitoxin system dominates the antitoxin. This causes dormancy, allowing persisters to escape antibiotics and other environmental factors that might kill growing cells<sup><a href="#fn6" id="ref6">6</a></sup>. | ||
Revision as of 11:07, 8 October 2016
Regulation of toxin expression
A problem that is encountered when expressing Bt toxins is that overexpression can be lethal to the bacterial chassis, in our case Escherichia coli. We aimed to prevent premature lysis of BeeT and achieve higher toxin yield by separating the growth phase from the toxin-producing phase. Toxin expression in coupled to quorum-sensing: only when a sufficient number of bacteria are present, the toxin is produced. Furthermore, we aimed to create a subpopulation of bacteria that do not produce the toxin, even when the bacterial density is high. When the toxin-producing bacteria perish, the subpopulation survives. As the survivors are genetically identical to the rest of the population, they are able to initiate a new growth phase and subsequently new toxin production.
The high toxin expression is needed to induce significant damage to the Varroa population. It is known that longer-term, low-dose exposure of a pesticide to the target organism can cause resistance (Tabashnik, Brévault & Carrière, 2013). Ideally, the toxin is only expressed when the target organism is present. To further optimize toxin expression, two measures to regulate expression were explored. BeeT was designed to sense the presence of Varroa destructor through the use of two riboswitches: one senses guanine, the other senses vitamin B12. Both are normally not present in beehives, but indicate the presence of the mite as guanine is a major component of mite faeces and B12 is present in the haemolymph of the bees. When a mite attaches itself to a bee to feed on the haemolymph, BeeT will be able to sense it.
Lastly, a toggle switch for toxin expression was designed, by combining the riboswitch and part of the light killswitch employed in our safety system (hyperlink). A hybrid promoter was designed that both facilitates continuous toxin production after transient sensing of the mite, and shuts off production when BeeT escapes from the hive and is exposed to light.
Detecting Mites
YOUR TEXT HERE
Quorum Sensing
Population dynamics
Overhauling population dynamics to improve toxin production
In our quest to save the honey bees from Varroa destructor we envisioned using Bacillus thuringiensis cry toxins. When activated and concentrated near the surface of cell membranes, Bt-toxins form pores inside the membrane, lysing the cell as a result1. High level constitutive Bt-endotoxin expression in Escherichia coli is known to inhibit growth and possibly kill the producing cells2. The aim behind the population dynamics subproject is to deliver a system for toxin regulation where production does not impair growth nor population survival of the bacteria.
Solutions to the problem of toxic expression are found in mechanisms that regulate protein production to minimize the negative effects on growth and survival3. Inducible expression for instance, is widely used to manually confine toxin expression in time to after the exponential growth phase of bacteria. We propose a regulation system that enables E. coli to separate the growth and the production phase by only expressing recombinant proteins when bacterial cell density is high. We cannot expect beekeepers to measure bacterial growth and induce expression themselves when the time is right.
Quorum Sensing: regulation based on bacterial density
For bacteria to regulate toxin production based on the density of bacteria, they need to be able to communicate bacterial density among them. Systems that enable bacteria to do so are called quorum sensing mechanisms. We have adopted one of the best known quorum sensing systems: the lux system originating form Vibrio fischeri. This systems consists of luxI and luxR two genes that allow for bacterial density communication. The LUXI protein is a synthase that produces acyl-homoserine lactones (AHLs). AHLs are small compound that diffuse across cell-membranes and function as autoinducers, the molecules that bacteria secrete to signal population density. A single cell produces insufficient autoinducer molecules to start quorum sensing. When there is a high density of bacteria, all producing AHL, the AHL concentration in the growth medium increases. AHL can then reach high enough cytoplasmic concentrations to effectively bind the LUXR protein. LUXR is a cytoplasmic receptor protein binding AHLs, regulating gene expression depending on whether AHL molecules are bound.To test density dependent expression, a two plasmid quorum sensing system was assembled in and transformed to E. coli DH5alpha cells. One plasmid contains the actual quorum sensing system BBa_K546000 in the pSB4K5 backbone, while the other plasmid contains a newly made GFP quorum sensing reporter BBa_K1913014 in the pSB1C3 backbone. Bacteria that contain both plasmids should be able to communicate their cell density and produce GFP once cell density gets high enough. The bacteria were indeed found to color green in overnight liquid cultures, preliminary indicating functional quorum sensing and reporting hereof (figure 3). The cells that contain only the reporter plasmid do not give a green pellet, indicating that they do not substantially activate expression of GFP.
Figure 3. Cell pellets of 10ml LB cultures of (left) DH5alpha cells containing both the quorum sensing system and the reporter plasmid. (right) DH5alpha cells containing only the reporter plasmid.
To follow the dynamics of the quorum sensing, plate reader measurements were done for the previously mentioned 2 plasmid quorum sensing E. coli. Fluorescence intensity was assessed by exciting at 480 nm and measuring light intensity of 510 nm. In addition, the optical density (absorbance at 600nm) was measured to relate fluorescence intensity to cell density (figure 4). Fluorescence over OD600 is plotted to correct the fluorescence intensity for the amount of cells that produce the fluorescence. Starting from an OD600 of 0.8 there is a sharp increase in fluorescence intensity per cell for the quorum sensing system. A very small increase in fluorescence/OD600 is observed for the reporter only control cells as well.
figure 4. Fluorescence and absorbance data for E. coli DH5alpha growing overnight. Continuous line: cells containing both the quorum sensing system and a GFP quorum sensing reporter. Dashed line: cells containing only the reporter. For both strains every value displayed is the average of at least three technical replicates and for each, three biological repeats were measured and plotted (the negative control lines show great overlap).
Subpopulation formation
Of course, population wide synchronized toxin overexpression is likely to kill all E. coli cells in the first wave of toxin expression. It would be more beneficial to increase the survival chances of some bacteria by keeping them as non-producers. Cells surviving the production phase would be able to initiate a new growth phase after death of the producing cells. The critical requirement for this is that while most cells display a certain behaviour (toxin production), some cells in the same population behave differently from the rest. A collection of cells that acts differently from the rest of the population is called a subpopulation.We hypothesized that a balance between two antagonistic genes can function as the basis for a subpopulation inducing circuit. The working of the dominantly expressed gene determines cell behaviour. We based our system on a modified lambda Prm promoter. This promoter is regulated by two cI ‘repressor’ proteins that have opposite effects on transcription of downstream genes. Despite its name, the cI repressor protein from phage λ induces the promoter, whereas the cI repressor protein from phage 434 represses the promoter. These two phage derived genes thus work antagonistically and function as the basis of the subpopulation system reported here. To actually create a subpopulation it is essential that the levels of the two cI proteins (or at least the ratios between them) can differ from cell to cell. Inspired by persister cell formation, we investigated expressing the two cI genes in one operon behind the same promoter. Difference in the turnover of the two proteins assures that changes in promoter strength induce temporary changes in the ratio between the level of the proteins. Imagine a system where both cI genes are transcribed at intermediate levels. 434 cI has a much stronger RBS than the λ cI gene and therefore is translated at a higher rate. This leads to a situation where there are more 434 cI than λ cI proteins: 434 cI is dominant. When the promoter regulating both genes is repressed, both genes will in time reach a new (lower) balance of protein levels. However, when 434 cI is degraded faster than λ cI, 434 cI protein levels will decrease more rapidly. Given the right tuning, this allows λ cI to briefly dominate the system. We hypothesize that small differences (cell age, metabolism etc.) between cells can determine whether λ cI indeed gets the chance to dominate the system. This principle of protein balance chances through changes in promoter strength is similar to how some cells become persister cells, while other continue growing5. We decided against an attempt to create actual persisters out of safety reasons. Persisters cells are a well studied yet poorly understood example of microbial subpopulations6. Persister cells make up around 1% of the population in the stationary phase7. In the main model for persister cells, persisters differ from other cells in the balance between toxin and antitoxin. In persister cells, the toxin in a toxin/antitoxin system dominates the antitoxin. This causes dormancy, allowing persisters to escape antibiotics and other environmental factors that might kill growing cells6.
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