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 RBS [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 [http://partsregistry.org/Part:BBa_G00100 VF2 primer] and a modified [http://partsregistry.org/Part:BBa_K237007 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

Testing

To test if our 4-brick inserted into E.coli it functioning, we have prepared 3 different systems: gene expression by measuring mRNA expression, MALDI to measure protein expression and a actual test of quorum quenching. The system is testing at different levels, and we thereby hope to be able to back-track the error if some of the systems aint working properly.

RNA expression

To measure if RNA is beeing expressed the are using northern blotting (See protocol). We are using a radioactive probe (32P) that binds to RIP mRNA. The probe is added to a membrane in which the RNA i run size- wise, and the probe is binding to the RNA containing the RIP sequence. The membrane it put on a film, that can store the radioactive phosphor, the film is scanned and we can thereby visualize if any accumulation of radioactive phosphor has taken place.

Test of quorum quenching

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 24 hours of growing the wells are washed three times with sterile 1xPBS to remove planktonic bacteria. Cells remaining adhered to the wells are subsequently stained with violet blue, the solution stands for 15 minutes and is again washed twice. A mixture of ethyl alcohol and acetone is added to each stained well to losen the biofilm, the biofilm is tested by measuring the absorbance by transfering 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 595 nm.

MALDI

Matrix-assisted laser desorption/ionization (MALDI) is a technique used in mass spectrometry, allowing the analysis of for example proteins.

Results

We have succeeded in assembling a 4-brick part containing the constitutive promotor with RBS, RIP and the terminator. This part has been sequenced and inserted into E. coli. The expression of RIP has been tested with Northern blot analysis, which confirmed that the gene is been translated to mRNA.

We are currently testing the quorum-quenching effect on S. aureus biofilm formation and the results are promising. We will continue these tests till we have conclusive results.

We have also submitted a part consisting of RIP and our chosen terminator. This parts has also been sequenced and is similar to the last part of our 4 brick. It is therefore possible to assemble this part with different kinds of promotors depending on what kind of transcriptional regulation is desired. Besides from the constitutive promotor we have also considered using a inducible promotor.