Revision as of 02:44, 3 October 2013

Bdh2 (intracellular)

This part expresses Bdh2 3HB dehydrogenase enzyme intracellularly. There is a GFP in operon with the enzyme which can be used to monitor expression levels. The promoter is arabinose inducible pBAD.

http://www.igem.org/wiki/images/f/f0/Reaction_bdh.jpg

Usage and Biology

bdh2 improves growth on 3HB

We have previously observed that MG1655 cells can survive in minimal media that contains 3HB as sole carbon source. This is evidence for that some uptake mechanism and a metabolic pathway is active at a low level in the cells. We hypothesised that the 3HB dehydrogenase could improve growth since it can convert 3HB into acetoacetate, a common metabolite in E.coli.

In order to test this, we run a growth experiment with various 3HB concentrations where the growth of cells containing bdh2 (BBa_K1149050) and Empty vector was recorded on a 96 well plate. We have calculated the growth rates from this data and plot it on the graph below.

3HB as a sole carbon sourcein M9 supplemented minimal media

Bdh2 with no pelB secretion tag growth. Bdh2 MG1655 cells were grown in M9S media to gauge growth, the growth does not differ from the control as p = 0.5543. Data points show final time point after 6h growth for each concentration. Growth was at 37°C with shaking over 6h. Error bars are SEM, n=4. Figure made by Imperial College London 2013 iGEM.

Bdh2 with no pelB secretion tag fluorescence. MG1655 cells with bdh2 were grown in M9S and then tested for fluorescence by measuring their GFP output. There is significant fluorescence as p < 0.0001. Data points show final time point after 6h growth for each concentration. Growth was at 37°C with shaking over 6h. Error bars are SEM, n=4. Figure made by Imperial College London 2013 iGEM.

Conclusion:We can conclude that there is a slight increase in the growth rate of bdh2 containing cells at 10 000 uM 3HB. This suggests that bdh2 is able to function as expected and produces acetoacetate which is used by the cell`s central metabolic pathways for growth. At 100uM or 100 000 uM, we did not observe any differences in growth. This could be be because the rate limiting step at low concentrations could be the uptake of 3HB rather than it`s conversion to acetoacetate. We hope that we could observe an increase in growth if we added the putative permease we designed to the system. At higher level, there is a drop in growth rates in both bdh2 and control cells, probably because of toxicity issues. In addition, fluorescence is highly elevated in bdh2 compared to empty vector control.

Growth Curve

Our bdh2 (intracellular) construct contains sfGFP within an operon and therefore fluorescence can be utilised to determine if expression is being induced by addition of Arabinose.

Bdh2 with no pelB secretion tag growth assay. MG1655 with intracellular bdh2 were grown over 6h to gauge the effect on growth when induced. Graph shows that while they reach the same end point, growth is initially faster without induction for BBa_K1149050, though this was not significant as a two-tailed t-test gave p = 0.4930 > 0.05, allowing us to accept the null hypothesis. Growth was at 37°C in LB with shaking. Error bars are SEM, n=4. Figure made by Imperial College London 2013 iGEM.

Bdh2 with no pelB secretion tag induction assay. In addition to testing BBa_K1149050 growth, we looked at induction whereby we see that more sfGFP is produced when induced. Although the promoter is leaky as the curve of intracellular bdh2, as seen with bdh2-pelB is followed tightly by the non-induced cells. Growth was at 37°C in LB with shaking. Error bars are SEM, n=4. Figure made by Imperial College London 2013 iGEM.

Conclusion: There is no growth inhibition caused by induction, nor is there any significant fluorescence induction.

Growth in Minimal media

Arabinose induction in M9 minimal media

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 [http://2013.igem.org/Team:Imperial_College/Protocols#Waste_Conditioned_Media_.28WCM.29 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.

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

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.

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.

Living Art

In addition to this, we have shown that bacterial paintings are achievable with this amazing biobrick (yellow):

References:

Sequence and Features