Our team found the component J23100-deGFP (BBa_K2205002) registered by iGEM17_Newcastle in the Parts, but the synthesis rate of deGFP in the Cell-Free system was lower than that in a cell-based system.
As a result, according to promoters and RBS suitable for Cell-Free systems reported in literature , through experiments and modeling, we found the combination of promoter and RBS that best expresses deGFP, and we registered it as BBa_K3885311 .
Usage and Biology
An E. coli cytoplasmic extract, absent any remaining living E. coli cells, provides the Transcription-Translation(TX-TL) molecular machineries. One of the unique features of this platform is at the level of transcription. Unlike conventional hybrid bacteriophage systems, transcription is solely based on the endogenous core RNA polymerase with the primary σ70.
Transcription is based on the core RNA polymerase and the primary sigma factor σ70 present in the cytoplasmic extract. All of the circuitries start with σ70 specific promoters.
The promoter P70a: previously described as P70, originates from the lambda phage repressor Cro promoter with the two operators sites OR2 and OR1 overlapping the −10 and −35 sequences. This σ70 E. coli promoter is the strongest so far reported. Unless otherwise stated, plasmids contain the untranslated region UTR1 from bacteriophage T7, one of the strongest bacterial untranslated regions, routinely used in recombinant protein expression.
The untranslated region (UTR), which contains ribosome binding sites, is also critical for regulating protein expression intensity. The strongest E. coli UTR reported to date is the main sequence of the g10 gene from phage T7, which we have named UTR1. When P70a is associated with UTR1, eGFP synthesis can reach up to 2 mg /mL(80μM) in batch mode reaction, comparable to using the T7 promoter.
deGFP：eGFP truncated and modified in N and C terminus
1.The expression of P70a-UTR1-deGFP
Kinetics of deGFP expression
Endpoints of deGFP expression
By using 12μL of reagent including 5nM of the plasmid P70a-deGFP, more than 70μM deGFP can be produced after 6 hours.
2.The synthetic rate of plasmid P70a-UTR1-deGFP at different concentration in the Cell-Free system.
3.The mutation of P70a and UTR1.
We have constructed these mutants: P70a-UTR1-deGFP(this part), P70a-UTR2-deGFP(BBa_K3885312), P70a-UTR3-deGFP(BBa_K3885313), P70b-UTR1-deGFP(BBa_K3885321), P70b-UTR2-deGFP(BBa_K3885322), P70b-UTR3-deGFP(BBa_K3885323), P70c-UTR1-deGFP(BBa_K3885331), P70c-UTR2-deGFP(BBa_K3885332), P70c-UTR3-deGFP(BBa_K3885333)。
Shin J, Noireaux V. Efficient cell-free expression with the endogenous E. Coli RNA polymerase and sigma factor 70. J Biol Eng. 2010 Jun 24;4:8.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000COMPATIBLE WITH RFC