Team:Michigan/Project
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===Results to Date=== | ===Results to Date=== | ||
We were able to put FimB onto pBAD, an arabinose inducible plasmid. This should have resulted in hyperpiliated E. coli, however we were not able to find many pili on our cells when we used an electron microscope. This is discouraging, but it's not a strong indication of failure because our growth conditions may not have been ideal for pili formation. More tests should be done to determine the optimal pili growth conditions. | We were able to put FimB onto pBAD, an arabinose inducible plasmid. This should have resulted in hyperpiliated E. coli, however we were not able to find many pili on our cells when we used an electron microscope. This is discouraging, but it's not a strong indication of failure because our growth conditions may not have been ideal for pili formation. More tests should be done to determine the optimal pili growth conditions. | ||
- | [[Image: OD_assay_all_samples.png | thumb | 500px|The results of an assay done on [[Team:Michigan/Pili_October#10/19/2010|10/19]]. ]] | + | [[Image: OD_assay_all_samples.png | thumb | 500px|Fig. 5 The results of an assay done on [[Team:Michigan/Pili_October#10/19/2010|10/19]]. ]] |
- | After conducting several assays to determine the effect of hyperpiliated E. coli on the flocculation of algae, we were not able to see very significant results. Hyperpiliated E. coli seem to flocculate the algae faster than nonpiliated E. coli, but the algae seemed to flocculate by itself. This is possibly due to contaminants in the algae. The algae was not grown in sterile conditions. | + | After conducting several assays to determine the effect of hyperpiliated E. coli on the flocculation of algae, we were not able to see very significant results (Fig. 5). Hyperpiliated E. coli seem to flocculate the algae faster than nonpiliated E. coli, but the algae seemed to flocculate by itself. This is possibly due to contaminants in the algae. The algae was not grown in sterile conditions. |
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The goals of the quorum sensing team are two-fold: first to characterize the response of ''E. coli'' to the ''C. vulgaris'' AI-2 mimic (which may be actual AI-2), and second to engineer ''E. coli'' to flocculate in response to the presence of ''C. vulgaris''. The first task will be accomplished by transforming a LuxS-mutant strain of ''E. coli'' (cannot produce its own AI-2) with an AI-2 reporter biobrick. We will then harvest supernatant from ''C. vulgaris'', which should contain AI-2 or its mimic, and apply it to the reporter strain to test its response. The second task will be accomplished by ligating a gene that causes over-expression of pili (characterized by the pili team) to the Lsr promoter, which is derepressed in response to AI-2. By transforming this part into LuxS-mutant ''E. coli'', we hope to create strain that will stick to algae and will flocculate only in the presence of ''C. vulgaris''. | The goals of the quorum sensing team are two-fold: first to characterize the response of ''E. coli'' to the ''C. vulgaris'' AI-2 mimic (which may be actual AI-2), and second to engineer ''E. coli'' to flocculate in response to the presence of ''C. vulgaris''. The first task will be accomplished by transforming a LuxS-mutant strain of ''E. coli'' (cannot produce its own AI-2) with an AI-2 reporter biobrick. We will then harvest supernatant from ''C. vulgaris'', which should contain AI-2 or its mimic, and apply it to the reporter strain to test its response. The second task will be accomplished by ligating a gene that causes over-expression of pili (characterized by the pili team) to the Lsr promoter, which is derepressed in response to AI-2. By transforming this part into LuxS-mutant ''E. coli'', we hope to create strain that will stick to algae and will flocculate only in the presence of ''C. vulgaris''. | ||
- | [[Image:QS_circuit.jpg|thumb|700px|left| | + | [[Image:QS_circuit.jpg|thumb|700px|left|Fig. 6 Regulatory mechanisms of the LsrR/phospho-AI-2 circuit in E. coli AI-2 uptake (modified from reference 70). The AI-2 uptake repressor |
LsrR represses many genes including the lsr operon (comprised of lsrACDBFG) and the lsrRK operon. AI-2 can be imported back inside the cell via | LsrR represses many genes including the lsr operon (comprised of lsrACDBFG) and the lsrRK operon. AI-2 can be imported back inside the cell via | ||
LsrACDB. Imported AI-2 is processed as phospho-AI-2 via the kinase LsrK. Phospho-AI-2 has been reported to bind LsrR and relieve its repression | LsrACDB. Imported AI-2 is processed as phospho-AI-2 via the kinase LsrK. Phospho-AI-2 has been reported to bind LsrR and relieve its repression | ||
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== Hy-Bi: Virus Protein Surface Display == | == Hy-Bi: Virus Protein Surface Display == | ||
- | [[Image:vp130ex.jpg|530px|left|thumb|Fig. | + | [[Image:vp130ex.jpg|530px|left|thumb|Fig.7 The binding of the viral surface protein on algae cells: (A) algae without the virus surface proteins; (B) and (C) algae with virus surface proteins]] |
The idea is to express algae-binding proteins on the surface of E. Coli in order to cause flocculation. Flocculation is the aggregation and precipitation of particles from solution. This flocculation will make the formation of biodiesel more efficient by eliminating the need for centrifugation to concentrate the fuel source. | The idea is to express algae-binding proteins on the surface of E. Coli in order to cause flocculation. Flocculation is the aggregation and precipitation of particles from solution. This flocculation will make the formation of biodiesel more efficient by eliminating the need for centrifugation to concentrate the fuel source. | ||
We devised two ways to cause flocculation, one being using a pili expression and the other using a virus surface protein, and the subject of our group is the virus surface protein. During its viral attack on cells, virus needs a protein that enables it to attach to the surface of the host cells. One of the virus surface proteins, vp 130, is used by chlorovirus to attach itself onto the surface of algae. Onimatsu, et al. recombined the vp 130 gene from Chlorovirus CVK2 with a plasmid, producing a wealth of vp 130. The binding of these proteins on their host cells, chlorella, was detected using fluorescent vp 130 specific antibodies (Fig.6)[12]. | We devised two ways to cause flocculation, one being using a pili expression and the other using a virus surface protein, and the subject of our group is the virus surface protein. During its viral attack on cells, virus needs a protein that enables it to attach to the surface of the host cells. One of the virus surface proteins, vp 130, is used by chlorovirus to attach itself onto the surface of algae. Onimatsu, et al. recombined the vp 130 gene from Chlorovirus CVK2 with a plasmid, producing a wealth of vp 130. The binding of these proteins on their host cells, chlorella, was detected using fluorescent vp 130 specific antibodies (Fig.6)[12]. | ||
- | [[Image:vp130.jpg|450px|left|thumb|Fig. | + | [[Image:vp130.jpg|450px|left|thumb|Fig.8 A model of vp 130-expressed bacteria binding algae cells.]] |
So, if we transfer the gene for this surface binding protein from the virus to E. Coli., each bacteria cell will bind to multiple algae cells, which bind to more bacteria, causing aggregation and flocculation. As opposed to using pili as the means to cause flocculation, the advantage of vp130 is its specific binding and aggregation of algae, automatically yielding a high algae mass in the flocculate (Fig.7). However, there are also disadvantages. For example, vp 130 is not a E. Coli surface protein, so it must be cloned as a fusion to a known surface protein. We also suspected OmpA and ice nucleation protein (INP) as candidate surface proteins. | So, if we transfer the gene for this surface binding protein from the virus to E. Coli., each bacteria cell will bind to multiple algae cells, which bind to more bacteria, causing aggregation and flocculation. As opposed to using pili as the means to cause flocculation, the advantage of vp130 is its specific binding and aggregation of algae, automatically yielding a high algae mass in the flocculate (Fig.7). However, there are also disadvantages. For example, vp 130 is not a E. Coli surface protein, so it must be cloned as a fusion to a known surface protein. We also suspected OmpA and ice nucleation protein (INP) as candidate surface proteins. | ||
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- | [[image:Ag43 colonies.gif|thumb|left| | + | [[image:Ag43 colonies.gif|thumb|left|Fig. 9 Colony morphology of cells examined by phase-contrast microscopy. Ag43-expressing strains containing pKKJ101 give rise to changes in colony morphology. |
Strains containing the control plasmid pUCP22 display the same morphotype as the parent strain. Colonies were grown overnight and photographed at the same | Strains containing the control plasmid pUCP22 display the same morphotype as the parent strain. Colonies were grown overnight and photographed at the same | ||
magnification. [13]|400px]] | magnification. [13]|400px]] | ||
- | [[Image:Ag43 Flocs.png|left|thumb| | + | [[Image:Ag43 Flocs.png|left|thumb|Fig. 10 Settling of cells from |
static liquid suspensions. (A) E. coli HEHA16(pKKJ101), (B) E. coli | static liquid suspensions. (A) E. coli HEHA16(pKKJ101), (B) E. coli | ||
HEHA16(pUCP22), (C) P. fluorescens SBW25(pKKJ101), (D) P. fluorescens | HEHA16(pUCP22), (C) P. fluorescens SBW25(pKKJ101), (D) P. fluorescens |
Latest revision as of 17:04, 27 October 2010