Team:Gifu/Experiments
Plasmid design
The design of DNA fragment (Seq.1) we used this year is shown below. In PURE system, T7 RNA polymerase is contained. Basically, we need to use T7 promoter designated by the inventors for transcription. SD sequence and Red fluorescent protein lie between BBa_K1859015 and BBa_K1859016. As terminator of this alignment, T7 terminator (BBa_K731721) was selected. In case of myTXTL, it uses σ70 promoter. When you use T7 promoter, you need to add T7 RNA polymerase. The recommended concentration of the polymerase is 2U/μL. SD sequence (RBS) and RBS are sandwiched by ribosomes. As T7 promoter, we did not use the sequence which has been registered in iGEM catalog. Instead of this, we used the new sequence of T7 promoter. RFP coding also contain TEV protease recognition site.
*Caution: BBa_K1332002 contains histidine tag. This time we excluded histidine tag. The function of the coding of this sequence is exactly the same as BBa_E1010 except for stop codon.
As proof of improvement of previous composite part of iGEM, BBa_K1491033 was provided by iGEM HQ. Typical alignment of T7 promoter is used in this part. However, in PURE system, the enhancer of the stem-loop structure need to be located in the downstream of T7 promoter to make an expression of protein intense. Thus we also design the part (Seq.2) below to show the improvement and this may contribute to the development of synthetic biology and iGEM competition. In Fig.1 we showed the detail of T7 promoter we used this year. Translation enhancer upstream of the SD sequence of mRNA, enhance the biosynthesis of protein. The enhancer may contribute to a direct interaction with ribosome protein S1. Fig.1 shows the detail of the enhancer.
*Caution: BBa_K1332002 contains histidine tag. This time we excluded histidine tag. The function of the coding of this sequence is exactly the same as BBa_E1010. In Seq.2 it has s stop codon.
Experiments
For activation of cell-free kit, both circular DNA and linear DNA is workable. We used linear DNA this time since we could not construct the plasmids. To amplify DNA fragment, PCR amplification or subcloning in E.coli is necessary. For the production, 0.5~3 ng/1 kb・μL of DNA for PURE system, 5 nM (Final concentration) of DNA for myTXTL was given to the reaction tube. Incubation time was typically 2 to 4 hours for PURE system, 15 hours for myTXTL. Observation of RFP under UV and SDS-PAGE was performed to confirm the expression. Flowchart of the cell-free expression is shown in Fig.2. You can perform PCR and cell-free expression in the same day. All DNA fragments were synthesized by IDT as gBlock fragment. The samples were sequenced and the alignments are all correct.
Cell free system
We used two types of cell-free system. The difference is shown in Table.1.
