Team:Aberdeen Scotland/Registry

From 2009.igem.org

University of Aberdeen iGEM 2009

BioBrick Experience

BBa_B0030

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Aberdeen_Scotland 2009

The miniprep, single and double digests all worked. However we did not use this part for further cloning.

BBa_B0034

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Aberdeen_Scotland 2009

Our miniprep, double digest and gel worked properly. We used this part in fusion PCR for building our biobrick BBa_K182102. The fusion PCR, cloning and sequence were all correct.

BBa_C0040

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Aberdeen_Scotland 2009

The transformation, miniprep and the gel (undigested and digested with the EcoRI and SpeI) worked as expected.

BBa_C0051

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Aberdeen_Scotland 2009

Our miniprep, double digest and single digest worked as expected. We used this part for further cloning which was successful. The sequencing was correct. However we can not confirm that this part was working as we did not test it's functionality.

BBa_E0840

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Aberdeen_Scotland 2009

Our miniprep, double and single digests worked as expected. We used this biobrick for building two of our parts (BBa_K182100 and 'BBa_K182101). We experienced very satisfactory cloning with high efficiency of transformants. The sequencing was correct. We discovered that all the bacteria containing this biobrick were releasing fluorescence even though there was no promoter upstream. The level of fluorescence observed was dependent on the plasmid copy number. We noticed that colonies displayed a visible, yellow-green colour, therefore selection was very easy.

BBa_I0462

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Aberdeen_Scotland 2009

The transformation, miniprep and digests, using XbaI and PstI, worked as expected. We did not test this part further, however, and can not confirm this parts functionality.

BBa_I13600

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Aberdeen_Scotland 2009

The gel analysis and the plasmid miniprep worked quite nicely as expected. But later the construct was further tested and characterised by combining with the bio-brick BBa_K182005 which contained a tet repressor under the control of a CI operator. So, the CFP expression of BBa_I13600was repressed as expected when tested with the BBa_K182005. With the addition of Anhydrotetracycline (an analogue of tetracycline) the CFP expression was restored and this was confirmed performing the fluorescent microscopy assay.



Expression can be visualised very nicely under a flurescent microscope. Even without having ideal filters. We used a GFP filter for the picture below.

2 I13600 flu60Aberdeen2009.jpg

Transforming BBa_K182005 into these cells should stop the production of CFP, since it expresses TetR. If it works as anticipated, the K182005 Tet repressor should bind to the Tet Operator of I13600 which would stop the expression of CFP.

1DanAberdeenigem2009fixed.png


Since K182005 is on an Ampicillin / chloramphenicol vector (pSB1AC3) and I13600 is on an Ampicillin vector, we decided to first transform I13600 into a cell and select for Ampicillin. We then checked for CFP expression and made the cells competent. After this we transformed K182005 into these cells and selected for Chloramphenicol.

With both plasmids now in the cell we checked for fluorescence, in the presence and absence of anhydrotetracycline (AHT or ATc), as can be seen in the picture below.

4 K182005-I13600 flu no AHTaberdeen 2009.jpg


16 K182005-I13600 no flu 1.8 AHTAberdeen2009.jpg


The above Pictures demonstrate that TetR effectivly binds to the I13600 Tet Operator.

BBa_I13601

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Aberdeen_Scotland 2009

The gel analysis and the plasmid miniprep worked quite nicely as expected. In addition, the construct was further tested by adding IPTG which took off the lacRepressor of the LacO and hence enabling the CFP expression.

BBa_I732100

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Aberdeen_Scotland 2009

The plasmid miniprep and the gel worked as expected. However, since we did not use this part for our cloning step we cannot comment on the sequencing or the other features of this part.

BBa_I732820

BBa_J37032

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Aberdeen_Scotland 2009

Experimental Design
The BBa_J37032 BioBrick expresses GFP under control of a LuxR responsive promoter. The BBa_J37032 part was provided by iGEM-HQ, was succesfully mini-preppred and transformed into E.coli. BBa_J37032 was used to test our constitutive LuxR/LuxI operon (BBa_K182200). For that purpose SCS1 E.coli was transformed either with BBa_K182200 alone (referred to as pIR in figure below), BBa_J37032 alone, or a combination of both plasmids. The single transformants acted as negative controls.

Results
It would be expected that the BBa_K182200 transformant would not fluoresce, since it does not express GFP. This was confirmed (top panel, figure below, showing light microscope pictures on the left, and fluorescent images on the right). Similarly, the single BBa_J37032 transformant was not expected to exhibit much fluorescence, since there is no endogenous expression of LuxR or LuxI in E.coli. A combination of BBa_K182200 and BBa_J37032 would be expected to produce high amounts of fluorescence at high cell densities as quorum sensing triggers the Lux promoter of BBa_J37032. However, in overnight stationary phase cultures at high density, it was found that the BBa_J37032 construct expresses the same high amount of GFP fluorescence with or without the presence of LuxR (compare middle panel [J37032 single transformant] with bottom panel [J37032/pIR double transformant])

Conclusion
We conclude that BBa_J37032 is either not responsive to LuxR, or that the pLux promoter is extremely leaky, driving significant GFP expression in the absence of LuxR. It therefore cannot easily be used to test quorum sensing due to this high background level of GFP expression.

J37TEST figure.jpg


BBa_K081008

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Aberdeen_Scotland 2009

Transformed OK and digested OK with enzymes EcoRI and SpeI.

BBa_R0040

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Aberdeen_Scotland 2009

The initial gel worked although the obvious confirmation was not possible due to the very small fragment size (54bp).But later it was confirmed following its usage by forming the part BBa_K182001 through the PCR gel analysis and the sequencing.The plasmid miniprep also worked.

BBa_R0051

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Aberdeen_Scotland 2009

The initial gel worked although the obvious confirmation was not possible due to the very small fragment size (49bp).But later it was confirmed following its usage by forming the part BBa_K182002 through the PCR gel analysis and the sequencing.The plasmid miniprep also worked.

BBa_R0062

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Aberdeen_Scotland 2009

Our miniprep, digest and gel gave expected results. However we did not use this part for our cloning.

BBa_S03518

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Aberdeen_Scotland 2009

The transformation, miniprep and the gel (undigested and digested with the EcoRI and SpeI) worked. Further testings were performed by the formation of the BBa_K182002, PCR analysis, sequencing and everything worked flawlessly, exceeding our expectations.

pSB1AC3

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Aberdeen_Scotland 2009

The transformation, miniprep and the gel (undigested and digested with the EcoRI and PstI) worked. Further testings were performed by the formation of the BBa_K182001 and BBa_K182005, PCR analysis, sequencing and everything worked as expected.

pSB1AT3

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Aberdeen_Scotland 2009

Plasmid rescued contained BBa_04450). The transformation, miniprep and the gel (undigested and digested with the EcoRI and PstI) worked. Further testings were performed through the PCR analysis and subsequent sequencing.

pSB3C5

Aberdeen_Scotland 2009

The transformation was twice unsuccessful. Plasmid rescued contained BBa_J04450.

pSB3T5

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Aberdeen_Scotland 2009

Plasmid rescued contained BBa_J04450. Transformation, miniprep, double and single digests all worked properly. It was noticeable that colonies were a light pink colour. We used this part for further cloning. This plasmid gave us a very high efficiency of cloning.

pSB4C5

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Aberdeen_Scotland 2009

Digestion with EcoRI and PstI worked well and complete digestion was observed. When pSB4C5-I52002 is transformed into XL1-blue cells, colonies are obtained. pSB4C5-I52002 carries the ccdB gene, toxic to wild-type E.coli strains. This plasmid was propagated in our experiments using the Invitrogen strain DB3.1, carrying the gyrA462 allele that confers resistance to this ccdB allele. During the generation of recombinant clones, the ccdB gene was excised and replaced by other BioBricks. For some of these experiments, E.coli strain XL1-Blue was employed. However, other iGEM teams should be aware that XL1-Blue also carries a gyrA allele, explaining our observation that this common E.coli strain was not killed by non-recombinant pSB14C5.

pSB4T5

Aberdeen_Scotland 2009

The transformation was twice unsuccessful. Plasmid rescued contained BBa_I52001.