Team:NCTU Formosa/results

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Results

The current progress of our project, including detailed information of the experimental data and the overall evaluation of the practicability of this project.

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RBS efficiency

We used the following biobrick to test the efficiency of different ribosome binding sites:

  1. Pcons+BBa_B0034+mRFP+Ter
  2. Pcons+[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1017202 BBa_K1017202]+mRFP+Ter
  3. Pcons+BBa_B0030+mRFP+Ter
  4. Pcons+BBa_B0032+mRFP+Ter
  5. control:pet40

As you can see from Fig 1, the plasmids of the biobricks above shows different expression of the reporter gene depending on the translational efficiency provided by the RBS employed. The redder the plasmid appears to be, the higher the florescence expression must be.

Fig 1. From left to right, the ribosome biding sites respectively: B0032, Target 1, B0030, B0034, control.
Fig 2. B0034 clearly expressed more RFP compared to others.


We measured the OD value along with the florescence expression of the reporter gene for each biobrick mentioned above. Fig 3 is the result obtained. We calculated the normalized expression of the y-axis by dividing fluorescence expression with the OD value measured, since the higher the OD value is, the larger the amount of bacteria that can express florescence would be. As shown in Fig 3, the normalized expression of the biobrick with B0034 is the highest and the one with target 1, K1017202, is second highest, while the other two of B0032 and B0030 show weak expressions. This result implies that target 1 can, in fact, serve as a functional RBS. In comparison to other RBS, target 1 can provide moderate translational efficiency that is just lower than that of highly efficient B0034 and higher than those of commonly used RBS such as B0030 and B0032.

Fig 3. Target 1 shows moderate expression compared to the others.

37 degrees Celsius RBS

Using biobrick, Pcons+37°C RBS+mGFP+J61048, we tested the function of the 37°C RBS at room temperature and at 37°C.

Fig 5. The biobrick for testing the function of 37°C RBS.
Fig 6. The differences between expressions of fluorescence can be easily observed under UV light.


As shown on Fig 7. the expression of mGFP under 37°C is much higher than the expression under 25°C. This graph demonstrate that 37°C RBS can effectively regulate gene expression by responding to temperature. The increased kinetic energy of 37°C is sufficient to causes the 37°C RBS to unfold and become available for ribosome binding. Under 37°C, for example at room temperature, the 37°C remain as hairpin structure that prevents ribosome binding. Such secondary structure cause the translational efficiency to be very low.

Fig 7. The normalized expression under 37°C is higher than the expression under room temperature by about 6 folds.



Expected sRNA regulation efficiency

To test the regulation efficiency of sRNA, we employed the following biobricks: Pcons + rRBS + mGFP + J61048 and Pcons + B0030 + lacI + J61048 + Plac + sRNA.

Fig 5. The biobrick of testing the sRNA.
Fig 6. sRNA regulation

The graph above shows the relationship between the concentration of IPTG added and the red florescent measured. The more IPTG added, the more sRNA can be produced by Plac as IPTG serves to activate the promoter. In other words, the amount of sRNA and the concentration of IPTG forms a liner relationship. With more IPTG, and therefore, more sRNA the amount of red fluorescent measured decreases. This implies that sRNA can effectively regulate the expression of RFP by decreasing the translation efficiency and the stability of rRBS.


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