Revision as of 20:40, 2 November 2014

mRNA circularization device (3� side)

This part consists of the 5' side of the intron(+exon fragment) from td gene of T4 phage(BBa_K1332004) and a double terminator(BBa_B0015). The protein coding sequence that is inserted between this device and mRNA circularization device (5’ side)(BBa_K1332008) can be circularized. But the 5' side of the intron(+exon fragment) from td gene of T4 phage contains a stop codon, so a circular mRNA made by this part doesn’t cause continuous translation.

explanation of this part

Figure 1. mRNA circularization device (3'side)(not endless translation)
Link:

• BBa_K1332004:The 5' side of the intron(+exon fragment) from td gene of T4 phage
• BBa_K1332005:The 3' side of the intron(+exon fragment) from td gene of T4 phage
• BBa_K1332008:mRNA circularization device (5´ side)
• BBa_K1332010:mRNA circularization device (3´ side)

Part which surrounded by red closing line is mRNA circularization device (3’side)(not endless translation).(figure 1)
This part has a stop codon, so a circular mRNA made by this part doesn’t cause continuous translation.

How to use

Insert the protein coding sequence between this device and mRNA circularization device (5’ side). How to use this part is same as how to use 5' side device(BBa_K1332008) or 3' side device(endless translation)(BBa_K1332009) . Thus,read that.

When protein coding is RFP coding, its results shows as follow.

Figure 2.In the case that RFP is inserted as protein coding.
Link:

• An RFP which we use is BBa_K1332002. That's an RFP which combines with a Histidine tag.
• BBa_K1332011:Histidine tag (8 AA) and RFP semi-permanent generator

Synthesis of long-chain proteins

Summary of the experiment

Confirm repeating translation by SDS-PAGE([http://2014.igem.org/Team:Gifu/Protocol#SDS protocol]).

Result

Figure 3. The result of SDS-PAGE
In the case that this part was used {Circular + RFP with stop codon (5’ intron)}, long-chain protein was not synthesized.

Sequence and Features