Team:NCTU Formosa/results

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 P_red biobrick for E. coli to respond to red light, both depicted in Figure 1. P_red is repressed in the dark and activated by red light.

To test whether red light can regulate P_red or not, we measured the florescence expression of E. coli that were exposed to red light under 37 °C. Figure 2 shows that red light can in fact activate P_red. 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.

Temperature-regulated System

37 °C RBS

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

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.

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. P_cons + BBa_B0034 + mRFP + Ter
2. P_cons + [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1017202 BBa_K1017202]+mRFP+Ter
3. P_cons + BBa_B0030 + mRFP + Ter
4. P_cons + 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.

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.

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 P_cons + 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.

Expected sRNA regulation efficiency

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

From Figure 14, it can be observed that sRNA can regulate RFP expression. The bacterial colonies without sRNA are red, showing high RFP expression. The bacterial colonies with sRNA, however, are white, suggesting that sRNA has regulated the RFP expression by base pairing to rRBS. The normalized expression without sRNA must be much higher than the expression with sRNA, or else we would not be able to easily distinguish the difference in RFP expression through the colors of the colonies.

Figure 15 shows quantitative analysis of the sRNA regulation efficiency. Colony A illustrates a high sRNA regulation efficiency compared to colony B. The reason that causes such difference in regulation efficiency is mutation. Colony B is mutated, causing the sRNA regulated-system to malfunction, while colony A is not mutated and shows a high regulation efficiency as anticipated.