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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Light-regulated System

Red Promoter

In order to positively regulate gene expression with red light, we employed the light receptor biobrick (K1017301) to give E. coli light sensing ability, and Pred biobrick for E. coli to respond to red light, both depicted in Figure 1. Pred is repressed in the dark and activated by red light.

Figure 1. Notice that Pred includes a 37 °C RBS that is only activated above or at 37 °C.

To test whether red light can regulate Pred or not, we measured the florescence expression of E. coli that were exposed to red light under 37 °C. Figure 3 shows that red light can in fact activate Pred. Colony 5 displays a high normalized expression under red light, suggesting that the biobrick was successfully activated by red light. On the other hand, colony 3 might be mutated, as it shows only little expression.

Figure 2. Positive control: Pcons + mGFP. Negative control: tet 30. Pred is activated by red light and shows strong GFP expression that is close to the expression of the positive control.
Figure 3.


Temperature-regulated System

37 °C RBS

Using biobrick, Pcons + 37 °C RBS + mGFP+J61048, we tested the function of the 37 °C RBS at 25 °C and at 37 °C.

Figure 4. The biobrick for testing the function of 37 °C RBS.
Figure 5. The differences between the level of GFP expressions can be easily observed under UV light.
Figure 6. The differences between the level of GFP expressions.

As shown in Figure 7 the normalized expression, obtained by dividing the florescence expression (emmision 612 nm and excitation 584 nm) with the OD600 value, of GFP under 37 °C is much higher than the expression under 25 °C. Such result demonstrates the fact that 37 °C RBS can effectively regulate gene expression by responding to temperature. The increased kinetic energy at 37 °C is sufficient to cause the 37 °C RBS to unfold and become available for ribosome binding. At 25 °C, however, there isn't sufficient kinetic energy to unfold the hairpin structure and the structure is preserved. As a result, the translational efficiency is very low at 25 °C.

Figure 7. The normalized expression (florescence expression/ OD value) under 37 °C is higher than the expression under room temperature by about 6 folds.



Small RNA-regulated System

rRBS efficiency

We used the following biobrick to test the translational efficiency of K1017202 (rRBS) compared to the efficiency of other RBSs:

  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: pet 30

As you can see from Figure 8, Figure 9, and Figure 10, the bacterial pellet and liquid of each biobrick shows different level of RFP expressions as the RBS of each biobrick provides a different translation efficiency. The deeper the red color is, the higher the level of expression is.

Figure 8. B0034 clearly expressed more RFP compared to others.
Figure 9. K1017202 shows moderate RFP expression that can be distinguied from the expressions B0030 and B0032
Figure 10. From left to right, the ribosome biding sites respectively: B0032, K1017202, B0030, B0034, control.

We measured the normalized expression for each biobrick mentioned above. We calculated the normalized expression 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 Figure 11, the normalized expression of the biobrick with B0034 is the highest and the one with K1017202 (rRBS) is the second highest, while the other two of B0032 and B0030 show weak expressions. This result implies that K1017202 can, in fact, serve as a functional RBS. In comparison to other RBS, K1017202 can provide moderate translational efficiency that is just lower than that of the highly efficient B0034.

Figure 11. K1017202 shows moderate expression compared to the others.

Effect on E. Coli Growth

To test whether or not our sRNA would effect the growth of E. Coli, we compared the growth of E. Coli with PSB1C3 (without RFP) and the E. Coli growth with Pcons + sRNA. It can be observed from Figure 12 that the resultant growth curves are similar, showing no signs of growth interference. This result proves that our sRNA regulated system can be integrated into bacteria.

Figure 12. The growth curve of the E. Coli with sRNA shows uninterrupted growth curve that is similar to the growth curve of the E. Coli with PSB1C3

Expected sRNA regulation efficiency

We employed the following biobricks to test the regulation efficiency of the sRNA we designed :
Pcons + rRBS + mRFP + J61048 and
Pcons + B0030 + lacI + J61048 + Plac + sRNA.

Figure 13. The biobrick of testing the sRNA.
Figure 14. The normalized expression decreases as the concentration of IPTG increases.

The concentration of IPTG added and the level of sRNA expression are in a linear relationship, since IPTG is the activator of lac promoter that regulate the expression of sRNA. With more IPTG added to raise the level of sRNA expression, the amount of green fluorescent measured decreases as graphed in Figure 14. This implies that sRNA can efficiently regulate gene expression through competitive mechanism. The more sRNA there is, the less ribosome binds to the mRNA to initiate translation.

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