Team:UNICAMP-Brazil/Coliguard/Recognition
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• How will our bacterial guards recognize the contaminants? | • How will our bacterial guards recognize the contaminants? | ||
- | Our idea is based on the premise that the engineered E. coli must be able to recognize contaminants in the culture medium as non-self. Thus, our bacteria need to recognize a specific signal that they don’t produce, but which are broadly produced or presented by potential contaminants. Thinking about the potential signals, we found two candidates: the AI2 (auto inducer 2) and the conjugation recognition mechanism. | + | Our idea is based on the premise that the engineered ''E. coli'' must be able to recognize contaminants in the culture medium as non-self. Thus, our bacteria need to recognize a specific signal that they don’t produce, but which are broadly produced or presented by potential contaminants. Thinking about the potential signals, we found two candidates: the AI2 (auto inducer 2) and the conjugation recognition mechanism. |
• Using AI-2 as a signal. How? | • Using AI-2 as a signal. How? | ||
- | As most bacterial species produces AI2 as a secondary metabolite, we decided to use this compound as a recognition factor. Our E. coli will be an AI2- strain and won´t produce native AI2. The presence of AI2 in the culture medium will then indicate the presence of contaminants, which will be recognized by an AI2 sensitive promoter present in our E. coli. This promoter will control the differentiation and killing mechanisms (see the links for more information). | + | As most bacterial species produces AI2 as a secondary metabolite, we decided to use this compound as a recognition factor. Our ''E. coli'' will be an AI2- strain and won´t produce native AI2. The presence of AI2 in the culture medium will then indicate the presence of contaminants, which will be recognized by an AI2 sensitive promoter present in our ''E. coli''. This promoter will control the differentiation and killing mechanisms (see the links for more information). |
• But, what if the contaminants do not produce AI-2? | • But, what if the contaminants do not produce AI-2? | ||
- | In these situations, our E.coli will use another recognition mechanism, based in conjugation. In conjugation, a donor bacterium only conjugates with organisms that don’t have the same conjugative plasmid. Bacteria that already have the plasmid display membrane proteins that prevent the conjugation with other bacteria that have the same conjugative plasmid. (reference) Therefore, if our E.coli has a conjugative plasmid, they will only conjugate with different organisms, which will be the contaminants. | + | In these situations, our ''E.coli'' will use another recognition mechanism, based in conjugation. In conjugation, a donor bacterium only conjugates with organisms that don’t have the same conjugative plasmid. Bacteria that already have the plasmid display membrane proteins that prevent the conjugation with other bacteria that have the same conjugative plasmid. (reference) Therefore, if our ''E.coli'' has a conjugative plasmid, they will only conjugate with different organisms, which will be the contaminants. |
- | Nevertheless, conjugation is a process that does not occur between E. coli and any other organism. There is a type of specificity to this process. A possible alternative to reduce this specificity is using multiple replication origins in the same conjugative plasmid (see Recognition by conjugation – lab strategy and results). However, this solution fails to address the whole problem. | + | Nevertheless, conjugation is a process that does not occur between ''E. coli'' and any other organism. There is a type of specificity to this process. A possible alternative to reduce this specificity is using multiple replication origins in the same conjugative plasmid (see Recognition by conjugation – lab strategy and results). However, this solution fails to address the whole problem. |
We think that a better way to improve the recognition system is to combine the conjugation signal with the AI-2 recognition mechanism. | We think that a better way to improve the recognition system is to combine the conjugation signal with the AI-2 recognition mechanism. | ||
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==Our recognition system: a combination of conjugation and the AI-2 recognition mechanism!== | ==Our recognition system: a combination of conjugation and the AI-2 recognition mechanism!== | ||
- | To improve our recognition system, we will use a signal produced at the beginning of the conjugation process in order to stimulate the production of AI-2 in our E.coli. This signal will induce the promoter pY, which controls the expression of the conjugation-related genes. This promoter will then control the expression of the AI-2 gene in our E.coli. As a result, when conjugation begins the promoter will be induced and thus cause an up-regulation in AI-2 gene expression. In this way, the conjugation process will activate the killing and differentiation mechanisms in a way that is AI-2-dependent. | + | To improve our recognition system, we will use a signal produced at the beginning of the conjugation process in order to stimulate the production of AI-2 in our ''E.coli''. This signal will induce the promoter pY, which controls the expression of the conjugation-related genes. This promoter will then control the expression of the ''AI-2'' gene in our ''E.coli''. As a result, when conjugation begins the promoter will be induced and thus cause an up-regulation in ''AI-2'' gene expression. In this way, the conjugation process will activate the killing and differentiation mechanisms in a way that is AI-2-dependent. |
Combining these two recognition systems, our ''E. coli'' will be able to recognize a vast group of contaminants. | Combining these two recognition systems, our ''E. coli'' will be able to recognize a vast group of contaminants. | ||
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As mentioned in the introduction, we will use conjugation as a signal to recognize contaminants. During conjugation, a donor bacterium only conjugates with organisms that don’t have the same conjugative plasmid. Bacteria that already have the plasmid display membrane proteins that prevent conjugation with other bacteria that have the same conjugative plasmid. | As mentioned in the introduction, we will use conjugation as a signal to recognize contaminants. During conjugation, a donor bacterium only conjugates with organisms that don’t have the same conjugative plasmid. Bacteria that already have the plasmid display membrane proteins that prevent conjugation with other bacteria that have the same conjugative plasmid. | ||
- | We chose the F plasmid to use in our project because it is the best-known conjugative plasmid. When looking for a recognition factor that could be used in our mechanism, scientific papers concerning the F plasmid mentioned the Py promoter, which controls the expression of conjugation-related genes in the tra-operon of the F-plasmid. This operon encodes the majority of the proteins involved in DNA transfer by conjugation in E.coli (reference). However, information concerning how the Py promoter works is lacking, and, therefore, we will need to conduct experiments to verify its action (see “Py promoter cloning and characterization” section). | + | We chose the F plasmid to use in our project because it is the best-known conjugative plasmid. When looking for a recognition factor that could be used in our mechanism, scientific papers concerning the F plasmid mentioned the ''Py'' promoter, which controls the expression of conjugation-related genes in the ''tra-operon'' of the F-plasmid. This operon encodes the majority of the proteins involved in DNA transfer by conjugation in ''E.coli'' (reference). However, information concerning how the ''Py'' promoter works is lacking, and, therefore, we will need to conduct experiments to verify its action (see “Py promoter cloning and characterization” section). |
- | Our idea is to use the Py promoter to act as a second signal (in addition to AI-2) to recognize contaminants, controlling the expression of the AI-2 gene and, consequently, the killing and the differentiation mechanisms that are AI-2-dependent. In order to do that, we first need to isolate the promoter and then, characterize its operation. | + | Our idea is to use the ''Py'' promoter to act as a second signal (in addition to AI-2) to recognize contaminants, controlling the expression of the ''AI-2'' gene and, consequently, the killing and the differentiation mechanisms that are AI-2-dependent. In order to do that, we first need to isolate the promoter and then, characterize its operation. |
- | In parallel, we will improve F plasmid to use in our engineered E. coli by, removing uninteresting parts in order to make it smaller and easier to copy. Moreover, we will remove the insertion sequences, which are the portions of the F plasmid DNA that mediate the integration into the bacterial chromosome, thus avoiding possible problems caused by F plasmid integration. (reference). Moreover, we will add different replication origins to the plasmid, thus increasing the range of contaminants that may be recognized (reference). | + | In parallel, we will improve F plasmid to use in our engineered ''E. coli'' by, removing uninteresting parts in order to make it smaller and easier to copy. Moreover, we will remove the insertion sequences, which are the portions of the F plasmid DNA that mediate the integration into the bacterial chromosome, thus avoiding possible problems caused by F plasmid integration. (reference). Moreover, we will add different replication origins to the plasmid, thus increasing the range of contaminants that may be recognized (reference). |
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'''Strategy''' | '''Strategy''' | ||
- | The strategy consists of PCR amplification of the Py promoter region by using primers that contain Biobrick prefix and Biobrick suffix, thus allowing us to clone the amplicon as Biobrick parts. Before designing primers, we verified that none of the restriction sites of the prefix or sufixe are contained in the amplified sequence. | + | The strategy consists of PCR amplification of the ''Py'' promoter region by using primers that contain Biobrick prefix and Biobrick suffix, thus allowing us to clone the amplicon as Biobrick parts. Before designing primers, we verified that none of the restriction sites of the prefix or sufixe are contained in the amplified sequence. |
What we have done | What we have done | ||
- | We designed primers from the F plasmid sequence (reference) to isolate the Py promoter by PCR. We used information on scientific literature to determine the region of the promoter (reference). We found that there is an intrinsic curvature, upstream of the promoter region, which could interfere in the promoter regulation. We decided, therefore, to design two pairs of primers: PyF1 - PyR1 and PyF2 - PyR1. These primer sets have the same reverse primers but different forward primers. The PyF1 – PyR1 amplicon has 133bp and includes the two closer bends upstream of the promoter region. The PyF2 - PyR1 amplicon has 75bp and doesn’t include any bend (see Notebooks – September 4th for more details). | + | We designed primers from the F plasmid sequence (reference) to isolate the ''Py'' promoter by PCR. We used information on scientific literature to determine the region of the promoter (reference). We found that there is an intrinsic curvature, upstream of the promoter region, which could interfere in the promoter regulation. We decided, therefore, to design two pairs of primers: PyF1 - PyR1 and PyF2 - PyR1. These primer sets have the same reverse primers but different forward primers. The PyF1 – PyR1 amplicon has 133bp and includes the two closer bends upstream of the promoter region. The PyF2 - PyR1 amplicon has 75bp and doesn’t include any bend (see Notebooks – September 4th for more details). |
We managed to amplify the pY promoter amplicons through PCR from the E. coli F plasmid, creating two new Biobrick parts: BBa_K284007 (PyF2 - PyR1 amplicon) and BBa_K284008 (PyF1 - PyR1 amplicon). | We managed to amplify the pY promoter amplicons through PCR from the E. coli F plasmid, creating two new Biobrick parts: BBa_K284007 (PyF2 - PyR1 amplicon) and BBa_K284008 (PyF1 - PyR1 amplicon). | ||
- | ==Py promoter cloning and characterization== | + | ==''Py'' promoter cloning and characterization== |
'''Strategy''' | '''Strategy''' | ||
- | After isolation of the Py promoter, it must be cloned in the BioBrick vector according to the standard assembly strategy. Afterwards, to characterize the promoter activity, we will make a device, with a reporter gene under the control of the Py promoter. We decided to use a fluorescent protein as a reporter gene for this characterization, such as the GFP (Green Fluorescent Protein). This reporter system will allow us to use the fluorescence signal in order to quantify promoter activity in a spectrofluorometer. (reference) | + | After isolation of the ''Py'' promoter, it must be cloned in the BioBrick vector according to the standard assembly strategy. Afterwards, to characterize the promoter activity, we will make a device, with a reporter gene under the control of the ''Py'' promoter. We decided to use a fluorescent protein as a reporter gene for this characterization, such as the GFP (Green Fluorescent Protein). This reporter system will allow us to use the fluorescence signal in order to quantify promoter activity in a spectrofluorometer. (reference) |
- | The characterization experiment consists of a conjugation experiment (reference) to be conducted using our engineered E. coli as the donor bacterium and another E. coli, without the F plasmid and containing a marker of resistance different from the F plasmid, as the receiver bacterium. After the beginning of the conjugation process, we will take samples after 1h, 2h, 3h, 6h, 9h, 12h and 24h. We will analyze these samples in a spectrofluorometer to quantify the fluorescence emitted. In addition, we will inoculate a portion of the sample in Petri dishes containing LB medium with the appropriated antibiotics to select only the receiver bacteria with the F plasmid. The plates will be incubated overnight at 37°C and then we will quantify the conjugation events through the observation of bacteria growing in the plates. Through this experiment, we will be able to analyze the Py promoter activity data in comparison with the conjugation events. If the measured Py promoter activity and the conjugation process begin coincide, we may conclude that the experiment worked and that the Py promoter can be used as a signal for conjugation and could thus be adapted into our recognition mechanism. | + | The characterization experiment consists of a conjugation experiment (reference) to be conducted using our engineered ''E. coli'' as the donor bacterium and another ''E. coli'', without the F plasmid and containing a marker of resistance different from the F plasmid, as the receiver bacterium. After the beginning of the conjugation process, we will take samples after 1h, 2h, 3h, 6h, 9h, 12h and 24h. We will analyze these samples in a spectrofluorometer to quantify the fluorescence emitted. In addition, we will inoculate a portion of the sample in Petri dishes containing LB medium with the appropriated antibiotics to select only the receiver bacteria with the F plasmid. The plates will be incubated overnight at 37°C and then we will quantify the conjugation events through the observation of bacteria growing in the plates. Through this experiment, we will be able to analyze the ''Py'' promoter activity data in comparison with the conjugation events. If the measured Py promoter activity and the conjugation process begin coincide, we may conclude that the experiment worked and that the ''Py'' promoter can be used as a signal for conjugation and could thus be adapted into our recognition mechanism. |
- | Literature reports that the Py promoter has a basal activity (reference). However, this fact won’t be a problem because its function will be adapted to the control of the production of AI-2 in the presence of contaminants. AI-2 is a type of signal that needs to exceed a threshold in order to stimulate the promoter sensitive to AI-2, which will be in turn coupled to the differentiation and the killing mechanisms. (reference). Therefore, the basal activity of the Py promoter should not be enough to trigger the differentiation and killing mechanisms. | + | Literature reports that the ''Py'' promoter has a basal activity (reference). However, this fact won’t be a problem because its function will be adapted to the control of the production of AI-2 in the presence of contaminants. AI-2 is a type of signal that needs to exceed a threshold in order to stimulate the promoter sensitive to AI-2, which will be in turn coupled to the differentiation and the killing mechanisms. (reference). Therefore, the basal activity of the ''Py'' promoter should not be enough to trigger the differentiation and killing mechanisms. |
What we have done | What we have done | ||
At first we intended to use the GFP as a reporter gene. We took the BBa_E0040 part, which contains the GFP, and started to work with it. Unfortunately, it didn’t work as expected. We did some restriction analyses and the bands expected after the electrophoresis running didn’t appear. | At first we intended to use the GFP as a reporter gene. We took the BBa_E0040 part, which contains the GFP, and started to work with it. Unfortunately, it didn’t work as expected. We did some restriction analyses and the bands expected after the electrophoresis running didn’t appear. | ||
- | After that, we decided to use another reporter gene. Searching for it in the Registry of Standard Parts, we found a device which is used to measure promoter activity, the exact construction that we need! So, we got this part (Bba_J23100) and, according to the standard assembly strategy, we substituted the promoter which already was there and put the Py promoter amplicons, resulting in BBa_K284008 (see Figure 1). We were only able to clone the PyF1 - PyR1 amplicon, the larger one, in this device, even after several repetitions. Therefore, we decided to continue experiments with this device only, in order to have time to perform the characterization experiment. | + | After that, we decided to use another reporter gene. Searching for it in the Registry of Standard Parts, we found a device which is used to measure promoter activity, the exact construction that we need! So, we got this part (Bba_J23100) and, according to the standard assembly strategy, we substituted the promoter which already was there and put the ''Py'' promoter amplicons, resulting in BBa_K284008 (see Figure 1). We were only able to clone the PyF1 - PyR1 amplicon, the larger one, in this device, even after several repetitions. Therefore, we decided to continue experiments with this device only, in order to have time to perform the characterization experiment. |
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As explained in the overview, another recognition mechanism is the AI-2 molecule itself. This process allows the identification of bacteria that are unable to conjugate, but able to produce the auto-inducer AI-2. | As explained in the overview, another recognition mechanism is the AI-2 molecule itself. This process allows the identification of bacteria that are unable to conjugate, but able to produce the auto-inducer AI-2. | ||
- | Therefore, we need a mechanism in which both recognition systems can be detected. In order to achieve that, the production of AI-2 has to be controlled by Py promoter. The LuxS gene, which encodes for the LuxS synthase, produce DPD (4,5-dihydroxy-2,3-pentanedione) that spontaneously forms the AI-2 molecule. This part was already designed by Davidson Missouri Western in 2008 and we will use it to produce AI-2 under the control of the Py promoter. In case of conjugation, Py will be activated and the LuxS synthase will be produced. On the other hand, microorganisms that naturally produce the AI-2 molecule skip that step. Once the recognition is achieved, it triggers the differentiation process through the expression of the CRE-recombinase. The Cre-recombinase will be under the control of the QseA promotor, which is activated in the presence of AI-2. The QseA also is known as the quorum-sensing E. coli regulator A and an array study showed that this regulator is regulated by AI-2 (7). The CRE-recombinase was already designed by Arkin Lab and will be utlized in this project. | + | Therefore, we need a mechanism in which both recognition systems can be detected. In order to achieve that, the production of AI-2 has to be controlled by ''Py'' promoter. The LuxS gene, which encodes for the LuxS synthase, produce DPD (4,5-dihydroxy-2,3-pentanedione) that spontaneously forms the AI-2 molecule. This part was already designed by Davidson Missouri Western in 2008 and we will use it to produce AI-2 under the control of the ''Py'' promoter. In case of conjugation, ''Py'' will be activated and the LuxS synthase will be produced. On the other hand, microorganisms that naturally produce the AI-2 molecule skip that step. Once the recognition is achieved, it triggers the differentiation process through the expression of the CRE-recombinase. The Cre-recombinase will be under the control of the ''QseA'' promotor, which is activated in the presence of AI-2. The ''QseA'' also is known as the quorum-sensing ''E. coli'' regulator A and an array study showed that this regulator is regulated by AI-2 (7). The CRE-recombinase was already designed by Arkin Lab and will be utlized in this project. |
Constructions: | Constructions: | ||
- | 1) BBa_K284027 - QseA promoter | + | 1) BBa_K284027 - ''QseA'' promoter |
- | This part will be constructed using the standard assembly strategy. After the amplification of the QseA promoter the part will be joined with the Bba_J23100 plasmid for characterization. | + | This part will be constructed using the standard assembly strategy. After the amplification of the ''QseA'' promoter the part will be joined with the Bba_J23100 plasmid for characterization. |
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- | 2) BBa_K284028 – LuxS under the control of Py promoter | + | 2) BBa_K284028 – LuxS under the control of ''Py'' promoter |
This device will be composed of BBa_K284008 (Py promoter), BBa_B0034 (a RBS), BBa_K091109 (LuxS) and BBa_0014 (a double terminator) and constructed following the standard assembly strategy. | This device will be composed of BBa_K284008 (Py promoter), BBa_B0034 (a RBS), BBa_K091109 (LuxS) and BBa_0014 (a double terminator) and constructed following the standard assembly strategy. | ||
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[[Image:Imagem3.jpg|center|400px]] | [[Image:Imagem3.jpg|center|400px]] | ||
- | 3) BBa_K284029 – CRE-recombinase under the control of QseA promoter | + | 3) BBa_K284029 – CRE-recombinase under the control of ''QseA'' promoter |
This device will be composed of BBa_K28402 (QseA promoter), BBa_B0034 (a RBS), BBa_J61047 (CRE-recombinase) and BBa_0014 (a double terminator) and constructed according to the standard assembly strategy. | This device will be composed of BBa_K28402 (QseA promoter), BBa_B0034 (a RBS), BBa_J61047 (CRE-recombinase) and BBa_0014 (a double terminator) and constructed according to the standard assembly strategy. |
Revision as of 19:32, 21 October 2009
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