Team:Hamburg/Part Description
Descripition
Gate
As a gate we used a toehold switch system which modulates a secondary structure in its mRNA that hides the ribosome binding site and the start codon of the gene that should be introduced in bacteria. Translation cannot occur due to hairpin formation. In figure 1 the loop structure of the mRNA is shown.
Figure 1: The scheme of a RNA based logic gate. Due to complementary regions the mRNA is forming a loop which hides the ribosome binding site and the start codon of the gene on interest. Our aim was to lock a chloramphenicol resistance behind that gate. Therefore we designed a plasmid with a strong promoter (J23102), our gate sequence, the chloramphenicol resistance, and a terminator (B1002). In figure 2 a scheme of this gate plasmid is shown.
Figure 2: The scheme of the gate plasmid. The sequence of the gene is flanked by a constitutive promoter (J23102) and a strong terminator (B1002). After the gate the gene for a chloramphenicol resistance is put.
Trigger
We designed two trigger which are forming a complex with a complementary region to a part of the gate. After binding the loop, the structure of the gate will open and release the ribosome binding site and the start codon. In figure 3 the mRNA structure of the trigger complex is shown.
Figure 3: The scheme of the trigger complex. Due to complementary regions in both trigger they form a T-like structure.
To transform the trigger into bacteria we put each of them on a plasmid with a promoter (J23100) and a terminator (B1002). In figure 4 the trigger plasmids are shown.
Figure 4: The scheme of the trigger plasmids. The sequences of the trigger is flanked by a constitutive promoter (J23100) and a strong terminator (B1002). After transformation of both trigger plasmids and the gate plasmid in one bacterium all three mRNA structures will be formed, the gate will be opened, and the translation of the chloramphenicol resistance can take place. Therefor bacteria which took up all three plasmids can be selected with chloramphenicol.
Gate/Trigger composition for testing To measure the functionality of our gate and trigger we designed a test plasmid which contains both trigger and the gate with GFP downstream of its sequence, each imbedded into a promoter and a terminator. Figure 5 shows the plasmid as we designed it. Figure 5: The scheme of the gate/trigger composition for testing. The testing plasmid contains both trigger, flanked by promoter and terminator, and the gate fused to GFP. Figure 6 shows how the trigger complex opens the gate due to complementary regions. After transformation of the plasmids into bacteria the fluorescence signal of GFP can be measured if all three parts work properly.
Figure 6: The scheme of the opening of the gate. Due to complementary regions the trigger complex binds to the gate, thus opens it. The ribosome binding site and the start codon are now accessible. Promoter composition for characterization Additionally, we wanted to test the strength of two promoters because we used them in our plasmids. To enable independent strength of a promoter it is necessary to insulate it from the following sequences. For this we inserted the ribozyme RiboJ (K1679038) after the promoter. Furthermore, we wanted to use GFP to measure the promoter strengths. For this we used the plasmid pSB1C3-BBa_E0240, from which we first had to remove a BsaI restriction site in the GFP sequence. Figure 7 shows both promoter test plasmids.
Figure 7: The scheme of the promoter plasmids for characterization with RiboJ. To measure the strength of both promoters (J23100 and J23102) the part E0240 (with GFP) was put behind them. The ribozyme RiboJ was used to insulate the promoter from the following part to enable independent expression strength.
Due to secondary structures in RiboJ we had problems to insert all parts into our backbone. Therefore we created plasmids containing only the promoters and BBa_E0240, without RiboJ. They are shown in Figure 8. Figure 8: The scheme of the promoter plasmids for characterization. To measure the strength of both promoters (J23100 and J23102) the part E0240 (with GFP) was put behind them.
