Endy:Screening plasmid/RNAseE notes
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RNA stability and independence
Two plasmids (I13513 and I13514) were constructed, one with GFP before the MCS and mRFP after, the other with the fluors reversed. First, the plasmids were characterized without a part in the MCS (specifically, without the ends capped and the MCS introduced). A significant difference was noticed between the two plasmids. When GFP was the first message on the transcript, the RFP signal was almost an order of magnitude stronger than when RFP was the first message. The GFP signal was nearly unchanged in the two cases.
It was hypothesized that a 5’ GFP was protecting the RFP message from RNAseE. Placing the RFP first would remove this protection and lower the RFP signal. GFP has been shown to be resistant to RNAseE cleavage, but no data exists for RFP. RNAseE is known to scan 5’ to 3’, so a resistant 5' sequence could protect a vulnerable mRNA.
If true, this presents a serious problem. Each part being characterized would affect the mRNA lifetimes of the fluors, changing the calibration. If the calibration depends on the identity of the part being measured, comparative measurements would be impossible. One solution was to add another promoter and avoid polycistronic messages if at all possible. However, this introduced another source of variation (two identical promoters might not produce identical POPs).
In an attempt to correct the problems associated with polycistronic messages, a technique was borrowed from Jay Keasling’s lab at Berkeley. Strong RNAseE sites were introduced upstream and downstream of the MCS, and a hairpin was placed 5’ of the mRFP1 transcript. There is no general RNAseE consensus sequence, so a naturally occuring site from the pap operon was chosen instead. This site was kept as short as possible to allow direct synthesis of the associated BioBrick parts. The hope was that the RNAseE sites would cleave the polycistronic message into two secondary transcripts and the 5’ hairpin would then protect the mRFP1 message from further degradation.
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