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Team:SDU-Denmark/Project
From 2009.igem.org
Forslag til opbygning:
1. Concept
2. Metode and results
3. Summary of results
4. Implications and development
Concept
The concept of our project is to assemble biobricks in to a plasmid and in this way get E. Coli to produce and excrete the quorum quenching protein RIP.
To do this we compose a plasmid containing a promoter, which should either be constitutive or inducible. After the promoter sequence we have a RBS, and then our RIP sequence, which we composted from the amino acid sequence of the protein and made in PCR via two costumed primers. We are working with a clean RIP sequence and a RIP sequence with an export sequence on. After the RIP sequence we have a terminator. It all has to be incorporated into an ampicillin resistant plasmid backbone.
We are trying with the two different promotors, because we don’t know whether a high concentration of RIP will quorum quench the E. Coli it self and it will there for be practical for us to control the rate of transcription. On the other hand we know that a higher concentration of RIP is more efficient at quorum quenching S. Aureus, and it will therefore be more efficient if the plasmid is transcribed at all times. Furthermore if the export of the protein out of the cell is weak, an overproduction of the protein might puncture the cell so RIP will leak out. This will of course kill the cell, but they could be replaced.
Methods
As seen in the flow chart, the parts RIP and sRIP are the first to be produce and ligated into backbone. Afterwards RIP and sRIP is ligated with a terminator resulting in a two brick. We are using a constitutive promoter that is already assembled with a terminator [http://partsregistry.org/wiki/index.php?title=Part:BBa_J13002 BBa_J13002] and a inducible promoter [http://partsregistry.org/wiki/index.php?title=Part:BBa_R0011 BBa_R0011] that we assemble with a RBS binding site [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 BBa_B0034] Finally the 4-bricks is constructed.
The steps was as following:
Interting parts into backbone: 1. amplification by PCR using VF2G01010 and a modified VR2 primer. (See protocol)
To skip a bacerial growth step, and thereby save some time, our parts where amplified by PCR, as suggested by the ginkogobioworks assembly manual [http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.pdf assembly manual]
2. Digesting with fast digest restriction enzymes EcoRI-HF™ and PstI (See protocol)
We experienced that digesting with slow digest buffers was ineffective and slow, why we replaced these with fast digest enzymes.
3. Ligate with T4 DNA Ligase (See protocol)
4. Transformation into competent E.coli (See protocol)
4. Testing length by colony-PCR (See protocol)
5. sequencing.
Ligation of parts:
1. amplification by PCR using VF2G01010 and a modified VR2 primer. (See protocol)
2. Digesting with fast digest restriction enzymes, upstream part with SpeI and downstream part with XbaI (See protocol)
3. Ligate with T4 DNA Ligase, resulting in a mixed site. (See protocol)
4. Interting parts into backbone
Test of quorum quenching mechanism
To test if our 4-brick inserted into E.coli it functioning and RIP is secreted out of the cell, we are designing a quorum quenching test system. The system is being made on a 4*6-well plade. Different S.aureus strains is growing in the wells making biofilms. Inhibition of biofilm formation is tested with different E.coli components added to the different wells: supernatant, crushed pellet and living E.coli. After one day of growing the wells are washed to remove planktonic bacteria. Cells remaining adhered to the wells are subsequently stained with violet blue. Ethanol is added to each stained well to losen the biofilm, the biofilm is tested by measuring the absorbance by transfering 125 μl of the crystal violet/ethanol solution from each well to a cuvette, and measure the optical density (OD) of each of these samples at a wavelength of 500 to 600 nm.
Results
