Regulatory
pBAD

Part:BBa_K206000:Experience

Designed by: Amelia Hardjasa   Group: iGEM09_British_Columbia   (2009-10-14)

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Applications of BBa_K206000

Imperial College London Experience


Growth in Minimal media

For our intracellular bdh2 construct, we used the pBAD strong promoter to induce.

Arabinose induction in M9 minimal media

450px-Arabinose_induction.png

Influence of Arabinose Concentration on growth in minimal media on bdh2. This showed that while growth was unaffected in bdh2 with Arabinose induction from 2-10 μM, M9WCM had decreased growth with an optimum at 10 μM. Characterisation was done in both M9 minimal media and M9 minimal waste conditioned media. Data points show final time point after 6h growth for each concentration. 0.4% glucose was used as a carbon source. Growth was at 37°C with shaking over 6h. Error bars are SEM, n=4. Figure made by Imperial College London 2013 iGEM.

450px-F_arabinose_induction.png

Influence of Arabinose Concentration on fluorescence in minimal media on bdh2. The trend shows that fluorescence of GFP in bdh2 is increased in M9 at lower induction concentration than M9WCM, however, the graph requires normalisation to better reflect reality. Characterisation was done in M9 and M9WCM. Data points show final time point after 6h growth for each concentration. 0.4% glucose was used as a carbon source. Growth was at 37°C with shaking over 6h. Error bars are SEM, n=4. Figure made by Imperial College London 2013 iGEM.

Conclusion: The null hypothesis, arabinose concentration does not influence growth of MG1655 in M9 minimal (M9M) media and M9 minimal waste conditioned media (M9MWCM) was tested using a two-tailed t-test. This must be rejected because p < 0.0217, thus arabinose concentration has an influence on growth in M9M and M9MWCM.

Glucose inhibition in M9 minimal media

450px-Glucose_inhibition.png

Glucose inhibition on growth in minimal media on bdh2. The outcome of this growth assay shows that cells grow more in M9WCM but that increasing glucose concentration influences growth in M9WCM while it is unaffected in M9 minimal media. Characterisation was done in M9 and M9WCM. Data points show final time point after 6h growth for each concentration. The arabinose concentration was kept at 6 μM for all samples. Growth was at 37°C with shaking over 6h. Error bars are SEM, n=4. Figure made by Imperial College London 2013 iGEM.

450px-F_glucose_inhibition.png

Glucose inhibition on fluorescence in minimal media on bdh2. The fluorescence of GFP in bdh2 under M9WCM shows significant difference in fluorescence output (p < 0.0001.). This compared to M9 media where fluorescence is almost half. The fluorescence in glucose is quite stable despite the decreased growth witnessed in the growth assay. Characterisation was done in M9 and M9WCM. Data points show final time point after 6h growth for each concentration. The arabinose concentration was kept at 6 μM for all samples. Growth was at 37°C with shaking over 6h. Error bars are SEM, n=4. Figure made by Imperial College London 2013 iGEM.

Conclusion: The null hypothesis, glucose concentration does not influence growth of MG1655 in M9 minimal (M9M) media and M9 minimal waste conditioned media (M9MWCM) was tested using a two-tailed t-test. This must be rejected because p < 0.0001, thus glucose concentration has an influence on growth in M9M and M9MWCM. In addition to this, the null hypothesis: glucose concentration does not influence fluorescence in M9 minimal media must be rejected as p < 0.0001. Thus glucose concentration influences fluorescence.



User Reviews

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Michigan iGEM 2012

According to sequence data, BBa_K206001 is missing O2 site necessary for repression by AraC. (Schleif R. AraC protein: a love-hate relationship. Bioessays 2003;25:274-282.)

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Imperial College London iGEM 2013

Promoter works, however expression can be detected in non-induced state.

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