Team:Newcastle/Parts

Parts

You can also find all the parts we've designed in the parts registry. We've used SBOL visual to specify our designs.

Part Information

YF1-FixJ Controlled 'Light Dependent Resistor'

We plan to engineer Escherichia coli to behave like a light dependent resistor. We aim to do this by using E. coli to vary the amount of free ions in an electrolyte in response to light. Ion uptake will be controlled by the expression of smtA. SmtA is a metallothionein that can bind to heavy metal ions like cadmium (II), Zinc (II) and Copper (II).

SmtA has been used in a number of iGEM projects and is in the registry (BBa_K519010). It has previously been used in experiments for Cadmium (II) uptake, see Tokyo-NokoGen 2011 and for accumulating Zinc (II) intracellularly. We will be examining firstly, the impact of smtA of Zinc (II) concertation rather than Cadmium (II) and then the impact that this has on the resistivity of the Zinc (II) containing media. In this instance we will be using Zinc sulfate (ZnSO₄) in solution where it disassociates into Zn²⁺ and SO₄^2- ions. Various concentrations of Zinc sulfate have known Electrical conductivity . When smtA is expressed it will render the Zn²⁺ unavailable and thereby reduce the conductivity of the solution.

We will be placing smtA under the control of a FixJ-P (phosphorylated FixJ) promoter. This allows it to be regulated by blue light through a series of reactions with its response regulator protein YF1 (below).

In the absence of light, YF1 undergoes autophosphorylation to produce YF1-P which can then phosphorylate FixJ. This in turn activates the transcription of the downstream protein, in this case it is SmtA. Thus, in the presence of light SmtA is not produced and so conductivity does not change, whilst in the absence of light SmtA is produced resulting in a decrease in resistance.

Clearly, this behaviour is the inverse of an electrical light dependent resistor where resistance increases with light intensity. To mimic this behaviour using biological circuits we would place an inverter before the FixK2 promoter (which is activated by FixJ-P). The inverter is constructed by placing the desired output, here SmtA, under the control of a lambda cl regulated promoter (BBa_R0051). As lambda cl represses the promoter having this produced under control of FixK2 promoter inverts the system so that SmtA is produced in the presence of light rather than the absence thereof. BBa_K592020 is an example of a part that uses this technique.

Our Construct

The non-inverted construct is shown in [FIGREF#1] and the inverted construct in [FIGREF#2]. Currently our device is shown as a 1 plasmid system but there is no reason that the two separate sub-components (constitutive expression of YF1 and FixJ, Fixj-P expression of SmtA) could not be split into a 2 plasmid system for easier assembly.

Figure 1: Standard Blue LDR construct.

Figure 2: Blue LDR with inverter.

For the non-inverted construct the parts are as follows:

BBa_J23100 - constitutive promoter

We will use a σ⁷⁰ constitutive promoter as this is the main E. coli sigma factor. Consequently, there should be RNA polymerase present to transcribe from this promoter at all stages during the bacterial growth cycle. Specifically, we have chosen BBa_J23100, an artificial promoter due to its widespread use, documentation and comparatively short sequence (35bp).

BBa_K592016 - FixJ & YF1 with RBSs

The YF1 and FixJ coding sequences are provided as a composite part together with standard RBSs in part BBa_K592016 which we have chosen for ease of assembly, in the event that we or future teams wish to use the BioBrick standard assembly to produce our part.

BBa_K592006 - FixK2

This is the wild-type promoter to which phosphorylated FixJ binds. It is reported that this promoter has very little leaky activity in the absence of FixJ.

BBa_K519010 - SmtA

This is the coding sequence for SmtA originally from Synechococcus sp, a cyanobacterial strain.

BBa_B1006 - Terminator

This is an artificial terminator part and was chosen because it has a high forward efficiency of 0.99.

pSB1C3 - Backbone

We are using the standard BioBrick backbone part pSB1C3 as this will make it easier to submit the part to the registry at a later date.

For the inverted part there are additional parts as follows:

BBa_C0051 - Lambda CI

This is the repressor protein from Lambda phage it represses the promoter BBa_R0051.

BBa_B0010 and BBa_B0012 - Double stop terminators

These are the terminators used in the composite part BBa_S04617 which is replicated in our construct.

BBa_R0051 - Lambda CI controlled promoter

This is a promoter from Lambda phage that is repressed by lambda Cl (BBa_C0051).

Construction

Synthesis

This construct can be sourced from IDT using our free allowance.

BioBrick Assembly

There exist a number of intermediate assembly components in the parts distribution that can be used to assemble our part faster if we use BioBrick assembly. Notably, BBa_S04617 contains the inverter,BBa_K592016 contains the FixJ and YF1. The two devices can be constructed separately as follows.

Constitutive Production Device

1. Cut the terminator BBa_B1006 with E & X.

2. Cut BBa_K592016 with E & S.

3. Mix & Ligate to form intermediate YF1FixJ+Terminator.

4. Cut the intermediate part with E & X and the constitutive promoter BBa_J23100 with E & S.

5. Mix and Ligate to form the constitutive production device.

SmtA Expression Device (inverted)

1. Cut BBa_K519010 with E & X.

2. Cut BBa_S04617 with E & S.

3. Mix and ligate to produce intermediate part: inverted smtA production.

4. Cut terminator with E & X.

5. Cut SmtA production intermediate with E & S.

6. Mix and ligate to produce SmtA expression device.

To produce the non-inverted device replace BBa_S04617 with the SmtA coding sequence and use an additional step to join this to an RBS, we suggest the standard RBS BBa_B0034 as this has good efficiency.

Biological 'Capacitor'

We plan to engineer Escherichia coli to mimic one of the properties of a capacitor, the ability to accumulate and hold charge for some time before discharging. This is shown in the idealised graph below.

An electrical capacitor accumulates charge whilst a voltage is applied and then discharges when the voltage stops being applied. We make an analogy between the voltage signal and protein concentration. Whereas an electrical capacitor accumulates charge, a biological ‘capacitor’ would accumulate proteins. Like an electrical capacitor which has a maximum charge it can accumulate, there is a maximum protein concentration that can accumulate in the cell determined by its production and degradation rate. Once proteins stop being accumulated in the cell it ‘discharges’ by having these drive the production of an output signal.

In this construct we use L-arabinose to mimic a voltage signal. This is entirely for experimental purposes, there is no reason that this device cannot be modified to respond to an electrical signal, for instance through the heat shock response explored elsewhere in our work. Although in this case we show that protein’s can be accumulated there is no reason why actual charge, in the form of a potential difference could not be generated across the cell membrane. There are already examples of membrane potentials in biology, the most obvious being found in neurons. This is something that has been explored by iGEM teams in the past e.g., Cambridge (2008). Importantly we show through modelling that the charge-discharge cycle can be mimicked in biological cells through the use of repressor/inducer competition. This could be merged with work on membrane potentials in the future.

Because we use a constitutively on promoter, the TetR repressible promoter (BBa_R0040) the default state of the system is ‘charging’. In this state lambda repressor (BBa_C1051) accumulates in the cell together with 434 repressor (BBa_C0052). The amount of 434 repressor grows faster than that of lambda repressor because there are two coding sequences for the protein in the circiut. This is to ensure that it outcompetes (on average) the lambda repressor so that there is a low output signal whilst in the charging state. This occurs because 434 repressor represses the output promoter whilst lambda repressor induces it. In out device the output signal is sfGFP (BBa_I746916).

We can switch the state of the system to the discharging state by causing the expression of TetR. To facilitate this we have used an L-arabinose promoter coupled with the TetR coding sequence to give us a chemical ‘off switch’. Once the TetR is produced the system enters the discharging state, no further protein synthesis in our construct is induced and so the amount of 434 repressor and lambda repressor start to decay. The 434 repressor is tagged with a very fast ssRA degradation tag, the LVA degradation tag so that it will be broken down faster than lambda repressor.

As this happens there will reach a point where the 434 repressor stops out-competing the lambda repressor and the output will start to be produced as it is induced by the lambda repressor. Whilst this is happening the level of 434 and lambda repressor will continue to fall until the output stops being produced and the system has completely ‘discharged’ and is in a resting state. At this point the removal of L-arabinose and the addition of tetracycline or an analogue thereof (which binds to TetR and prevents it from repressing the promoter) would switch the system back into the charging state and the process can begin again.

Construct

Our construct, as shown here, is a composite part which is built entirely of BioBricks already in the registry. The parts used are as follows.

Part No.	Name	Purpose
BBa_R0080	L-Arabinose Promoter	This part is an L-Arabinose inducible promoter with very low level expression in the absence of L-Arabinose and AraC. Be aware, if the E. coli strain used constitutively expresses AraC then this promoter will ‘leak’. Check the strain list for information.
BBa_R0040	TetR	This is the coding sequence for TetR which represses BBa_R0040.
BBa_R0040	TetR Repressible Promoter	This is a constitutively on promoter which can be repressed by TetR. Be aware, if the strain used expresses TetR constitutively then this promoter will be repressed. Check the strain list for information.
BBa_C0052	434 Repressor	Represses the output promoter.
BBa_C0051	Lambda Repressor	Induces the output promoter.
BBa_I12006	Modified Promoter Part	This is a modified promoter part, originally the lambda Prm promoter. The modification allows it to be activated by lambda repressor and repressed by 434 repressor.
BBa_I746916	Superfolder GFP	This is the coding sequence for super folder GFP. We have chosen to use this as our reporter because it can easily be quantified using a plate by taking the OD600 measurement. This is harder to quantify with more visible reporters like amilCP.
BBa_B1006	Standard Terminator	We chose to use this promoter from the registry as it has a high forward efficiency.
BBa_B0034	RBS	We chose to use this RBS from the registry as it is efficient and widely used in iGEM projects.

Adding parts to the registry

You can add parts to the Registry at our Add a Part to the Registry link.

We encourage teams to start completing documentation for their parts on the Registry as soon as you have it available. The sooner you put up your parts, the better you will remember all the details about your parts. Remember, you don't need to send us the DNA sample before you create an entry for a part on the Registry. (However, you do need to send us the DNA sample before the Jamboree. If you don't send us a DNA sample of a part, that part will not be eligible for awards and medal criteria.)

We encourage you to put up much more information as you gather it over the summer. If you have images, plots, characterization data and other information, please also put it up on the part page.

<groupparts>Newcastle</groupparts>