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| - | {{:Team:Newcastle/CSS}}
| + | #REDIRECT[[Team:Newcastle/Project]] |
| - | {{:Team:Newcastle/Header}}
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| - | {{:Team:Newcastle/Left}}
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| - | == Overall project ==
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| - | The aim of our project is to genetically engineer ''Bacillus subtilis'' to be able to detect and sense cadmium which has been taken up from the soil environment and sequester them into a metallothionein. This metallothionein will then become incorporated into a Bacillus spore; the resilience of which means that the cadmium ions can become isolated from the environment (and made bio-unavailable) for many years.
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| - | This project involves a number of steps, each of which can be considered as sub projects:
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| - | # Metal intake | + | |
| - | # Metal sensing
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| - | # Tuning of ''Bacillus subtilis'' normal stochastic switch
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| - | # Metal sequestration by metallothionein
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| - | # Second stochastic switch
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| - | # Synthesizing a Promoter Library
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| - | <br>
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| - | == Metal Intake ==
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| - | For this project we want to be able to process cadmium and cadmium only. Therefore it would be logical to find a way in which we can increase the intake of cadmium without increasing the intake of other metals too.
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| - | It is known that a ''Bacillus subtilis'' cell (from the 168 strain) takes up cadmium naturally through the '''manganese transport system''' <font color="gray">(Laddaga. R.A., Bessen. R., Silver. S.; 1985)</font>. Additionally it has been demonstrated that mutations to the manganese transport system can affect its ability to uptake cadmium without affecting its ability to transport manganese <font color="gray">(Zeigler. D.R., et al; 1987)</font>.
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| - | The manganese ion channel that we intend to either upregulate or control is the '''''mntH''''' ion channel (also goes under the name ydaR). mntH is part of the Nramp family of proton-coupled, metal ion transporters <font color="gray">(Que. Q., Helmann. J.D.; 2000)</font>. It is also classified as a secondary transporter (Membrane Transport website).
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| - | It is also known that mntH is regulated negatively by increasing manganese ion concentrations. This is allowed to happen through the promoter called '''mntR''' <font color="gray">(Que. Q., Helmann. J.D.; 2000)</font>. This means that B.subtilis has the ability to limit the manganese metal intake system when the intracellular concentrations of Mn+ starts to near cytotoxic levels.
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| - | == Metal Sensing ==
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| - | If our project is to process cadmium and not other metals, we need to genetically engineer ''Bacillus subtilis'' to carry out a set of cellular processes based on the action of metal sensors. These metal sensors will detect cadmium through a system known as '''AND Gating'''.
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| - | There are two metal sensing repressors, which are known to respond to cadmium: '''''arsR''''' and '''''czrA''''' (also known as '''''yozA''''').
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| - | ===='''<u>i) arsR</u>'''====
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| - | '''''arsR''''' (also known as '''''yqcJ''''') is a protein which is part of the ''ArsR-SmtB'' family of transcriptional regulators. It is a regulating protein for the '''arsenic resistance operon''' in ''Bacillus subtilis'' <font color="gray">(NCBI website – arsR family transcription regulator profile) (Moore. C.M and Helmann. J.D; 2005)</font>.
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| - | The ''arsR'' protein acts as a repressor until it is conformationally changed by the presence of '''arsenic ions''' <font color="gray">(Harvie. D.R, et al; 2006)</font>. However, arsenic is not the only metal which can cause this action to happen; it has been noted that '''silver (Ag(I)), cadmium (Cd)''' and '''copper (Cu)''' <font color="gray">(Moore. C.M and Helmann. J.D; 2005)</font> can cause this action to happen.
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| - | {| class="wikitable" border="1"
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| - | ! Metal Sensor
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| - | ! colspan="4" | Metals Sensed
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| - | | ''arsR''
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| - | | As(III)
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| - | | Ag(I)
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| - | | Cu
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| - | | Cd
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| - | |}
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| - | ===='''<u>ii) czrA (yozA)</u>'''====
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| - | '''''czrA''''' (also known as '''''yozA''''') is a member of the ''ArsR-SmtB'' family of transcriptional regulators <font color="gray">(Moore. C.M and Helmann. J.D; 2005)(Harvie. D.R, et al; 2006)</font>. Like ''arsR'', it is a regulator protein which can be relieved from binding to the DNA by being bound to by metal ions – these include '''zinc (Zn), cobalt (Co), nickel (Ni)''' and '''cadmium (Cd)''' <font color="gray">(Moore. C.M and Helmann. J.D; 2005)</font>. This can be summarised in the table below:
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| - | {| class="wikitable" border="1"
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| - | |-
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| - | ! Metal Sensor
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| - | ! colspan="4" | Metals Sensed
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| - | |-
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| - | | ''czrA''
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| - | | Zn
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| - | | Co
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| - | | Ni
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| - | | Cd
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| - | |}
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| - | ===='''<u>AND Gating</u>'''====
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| - | If the two ion selectivity tables are put together, it can be seen that the '''metal common to both sensors is cadmium''':
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| - | {| class="wikitable" border="1"
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| - | |-
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| - | ! Metal Sensor
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| - | ! colspan="4" | Metals Sensed
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| - | |-
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| - | | ''arsR''
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| - | | As(III)
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| - | | Ag(I)
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| - | | Cu
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| - | | bgcolor="yellow" | Cd
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| - | |-
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| - | | ''czrA''
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| - | | Zn
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| - | | Co
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| - | | Ni
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| - | | bgcolor="yellow" | Cd
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| - | |}
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| - | This means that if the two repressors can act on a single promoter or binding site by AND gating, cadmium can be detected and a biological response can be triggered.
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| - | == Tuning of ''Bacillus subtilis'' normal stochastic switch ==
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| - | == Metal sequestration by metallothionein==
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| - | So the cadmium has made its way into the cell; and the cadmium has been detected by the metal sensors <i>arsR</i> and <i>czrA</i>. The question is: what happens to the cadmium now?
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| - | In our project, we hope to soak up the intracellular cadmium with a metallothionein known as <i>smtA</i>. Metallothioneins are proteins which have great tendencies to bind to cationic metal ions; examples of which include copper (Cu), zinc (Zn), lead (Pb) and cadmium (Cd) <font color="gray">(Creti. P., et al; 2009)</font>. This property is due to the richness of cysteine residues in its structure <font color="gray">(Creti. P., et al; 2009)</font>.
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| - | In the cell, metallothioneins generally have two important roles: to remove non-essential metals via sequestration and to control levels of essential metals <font color="gray">(Creti. P., et al; 2009)</font>. It is the first role with which we are concentrating on.
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| - | <i>smtA</i> encodes a metallothionein of the same name – this protein, which is described as a class II metallothionein seems to be synthesized in response to metals such as zinc and cadmium <font color="gray">(Morby. A.P., et al; 1993)</font>. This suggests it has a role in cadmium sequestration.
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| - | We intend to coat our ''Bacillus'' spores in the metallothionein by making a fusion protein with ''cotC'' a major spore coat protein, our metal sponge should locate to the spore making the cadmium bio-unavailable.
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| - | == Second stochastic switch ==
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| - | Our second stochastic switch will use an invertible promoter sequence to regulate expression of either germination genes or genes required for our spores to be metal containers, such as ''smtA'' the metallothionein. We will carry out stochastic modelling of the system in order to fine tune it to produce a 'biased heads or tails' switch.
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| - | == References ==
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| - | Membrane Transport website (http://www.membranetransport.org/all_type_btab.php?oOID=bsub1 )
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| - | Charles, M. M., G. Ahmed, et al. (2005). "Genetic and physiological responses of Bacillus subtilis to metal ion stress." Molecular Microbiology 57(1): 27-40.
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| - | Cretì, P., F. Trinchella, et al. "Heavy metal bioaccumulation and metallothionein content in tissues of the sea bream Sparus aurata from three different fish farming systems." Environmental Monitoring and Assessment.
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| - | Duncan, R. H., A. Claudia, et al. (2006). "Predicting metals sensed by ArsR-SmtB repressors: allosteric interference by a non-effector metal." Molecular Microbiology 59(4): 1341-1356.
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| - | Laddaga, R. A., R. Bessen, et al. (1985). "Cadmium-resistant mutant of Bacillus subtilis 168 with reduced cadmium transport." J. Bacteriol. 162(3): 1106-1110.
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| - | Moore, C. M. and J. D. Helmann (2005). "Metal ion homeostasis in Bacillus subtilis." Current Opinion in Microbiology 8(2): 188-195.
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| - | Morby, A. P., J. S. Turner, et al. (1993). SmtB is a metal-dependent repressor of the cyanobacterial metallothionein gene smtA: identification of a Zn inhibited DNA-protein complex. 21: 921-925.
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| - | Qiang, Q. and D. H. John (2000). "Manganese homeostasis in Bacillus subtilis is regulated by MntR, a bifunctional regulator related to the diphtheria toxin repressor family of proteins." Molecular Microbiology 35(6): 1454-1468.
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| - | Zeigler, D. R., B. E. Burke, et al. (1987). "Genetic mapping of cadmium resistance mutations inBacillus subtilis." Current Microbiology 16(3): 163-165.
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| - | {{:Team:Newcastle/Footer}}
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| - | {{:Team:Newcastle/Right}}
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