Team:HKUSTers/Project

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The Design

"Making the dice"

We "tell" our cells to start making the "dice"--T7 polymease by applying a pulse of IPTG. IPTG can activate the araC/pBAD promoter which leads to transcription of T7 polymerase.

"Throwing the dice"

The core part of the randomizer consists of a pair of overlapping T7 promoters having similar binding affinities. The T7 polymerase binds randomly onto either the left or the right promoter to give an either 0 or 1 signal (reprenting by giving RFP or GFP).The binding is mutually exclusive, for if one binds to the promoter, it will block the binding of the other one as the two T7 promoters are overlapping to each other.

Reciprocal inhibiton

Then there are TetR and C1434 repressor repressor genes on both sides respectively. A repressor protein can be produced to interrupt transcription on the other side of the circuit. For example, say, the T7 polymerase binds onto the promoter on the left side. The TetR repressor on the left, which was coded by TetR repressor gene, will bind to TetR operator on the right and repress transcription of right side of the promoter. Thus, the intial random event (binding of polymease) can be captured and amplified in a postive feedback circuit.

Final outcome

Continous production of GFP if the polymease binds to the left of overlapped T7 promoter
Continous production of RFP if the polymease binds to the right of overlapped T7 promoter

The signal output is shown by green or red fluorescent protein (GFP/RFP) respectively.

Construction details

Testing

Testing of the components of circuit
Name of test	Purpose	Circuit involved
T7 polymease production test	Check the T7 polymease production circuit using IPTG as inducer and GFP as reporter
GFP test	Check if the intact T7 promoter and GFP is functional
Adjacent bidirectional promoters test	Evaluate if two promoters in opposite directions can function properly if they are placed adjacently
Truncated promoter test	Stimulate the condition in the overlapped version, where the first two non-essential base pairs of cl434 promoter are altered. We want to know if the cl434 can still function under such conditions.
Left/right induction test	To see whether the overlapping promoter can drive transcription of both directions

Strain and vector used

Strain: E. coli BBa_V1001 DH5a, with genotype

 F-φ80dlacZΔM15 Δ(lacZYA-argF)U169 deoR recA1 endA1 hsdR17(rk- mk+ phoA supE44 λ- thi-1 gyrA96 relA1)

Vector: pUC18+ λ-InCh2

Integration system

To ensure the randomness of the system we make, we should make sure there is only one copy of the circuit. Therefore, we are trying to incoporate the circuit into the chromosome.

We have chosen the λInCh system as the vehicle for intergration.

The success rate for recombination is about 1 in 1000 cells. Ampicillin is used to screen for the second recombination event and temperatute of 42 degree Celsius is used to screen for the third recombination event. (Such temperature deactivates the cl857 repressor and hence activates the harmful gene of λ-phage introduced in second recombination event, killing the host cells.)

The advantages of this approach include:

No risk of >1 copy of target gene in our machine, so as to give a sharp output instead of "blurred" output caused by multiple copies
Stable integration of a single copy of the construct DNA fragment
Most ordinary E. coli strains and a variety of pBR322-derived Amp-resistant plasmids can be used
λ-phage as the only specialized vector required

Source:

Boyd D., Weiss D.S., Chen J.C., Beckwith J., Towards single-copy gene expression systems making gene cloning physiologically relevant: Lambda InCh, a simple Escherichia coli plasmid- chromosome shuttle system (2000) Journal of Bacteriology, 182 (3), pp. 842-847.

Total construnct and detailed experiment steps

The Initial Proposal

The first draft of project description

The HKUSTers’ project is composed of three components; (1) a Randomizer with one of the two possible random outputs at a single cell level, (2) a Memorizer that specifically records the last input and compares it with the current input to give out an XOR calculation, while the input before the last is erased, and (3) a Spatial Oscillator that facilitates autonomous back and forth migration of cells responding to two opposing chemical gradients.

Our project objective is to construct a biological slot machine by coupling the Randomizer with the Memorizer. When two consecutive random signals are produced by the Randomizer, a Memorizer can evaluate whether the two signals are the same so as to display a Jackpot output. Coupling of the Randomizer and a Spatial Oscillator can generate a dynamic pattern, such as dots and stripes, whereas modifications of the Spatial Oscillator can be introduced to produce an automatic and more complex oscillation pattern. With these three building blocks integrated in specific manner, different dynamic patterns can be generated to recreate stripes and pigmented profiles as found in many biological systems. The components also constitute the foundation units subjected to scale up assembly of more complex operations.

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 ===Total construnct and detailed experiment steps===
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 ==The Initial Proposal==