Our initial idea to prove our concept of antitermination was to use flourescent proteins as reporters. This approach gives the opportunity to measure the termination and antitermination efficiency of our designed BioBricks in vivo as well as in vitro, the latter using a translation kit based on e.coli lysate.
We decided to use the flourescent proteins GFP and RFP, as their spectra do not overlap and we would not measure any FRET. We would use GFP fluorescence as internal control and RFP fluorescence as signal to detect termination/antitermination by our switch we cloned in between the coding sequences of the proteins. Both protein sequences are under the control of one (L-arabinose induced) promoter.

When measuring the termination of our BioBricks and the antitermination by their corresponding signal-RNA, we should be able to observe an increasing RFP emission compared to the GFP emission upon induced signal-RNA production in the cells/in the kit:

Wiith these measurements, it should also be possible to observe differences in efficiency of termination as well as antitermination between our designed switches.

Measurements with the malachite green aptamer as reporter

A second possibility to measure parameters of our switches we came up with, was the idea to investigate our system on the transcriptional level only. Therefore, we decided to use malachite green as reporter. Malachite green in a fluorescent dye, whose emission increasing dramaticly (about 3000 times) upon binding of a specific RNA-aptamer.

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To study the switches on the transcriptional level gives the advantage, that we would have less interferences and possible artefacts. Also, we are not sure how cellular mechanisms like degradation of RNases or interacting factors as well as molecular crowding influence our systems.

We made constructs comprising of a sigma(70)-binding promoter followed by a short nonsense sequence, the switches and the aptamer sequence.
Also we made constructs, where the transcription of the signal-RNA is under the control of a sigma(70) promoter. These two linear DNA-constructs, together with the e.coli RNA-polymerase and the right buffer conditions should represent an easy-to-handle measurement kit on the transcriptional level.

Results

Flourescent proteins

Unfortunatly, we had to change the reporter construct two times during our experiments as several problems occured in our measurements:

First Try: based on the measurement plasmid pSB1A10

At the beginning, we decided to use the reporter plasmid pSB1A10 from the registry. It consists of the fluorescent proteins eGFP and mRFP1. Each sequence includes a ribosome binding site and a stop-codon; the two genes are divided by a cloning side including the BioBrick cleavage sites.
In front of the eGFP sequence, the plasmid includes an arabinose-inducable promoter. The plasmid also contains an ampicilline resistence.

We cloned our switches into the cloning site of the measurement plasmid and used an empty cloning site as control; our signal-RNAs we cloned into the pSB1K3 vector, together with the BioBricks R0011 (Lac promoter) and B0014 (double terminator of transcription). Afterwards, we cut pSB1K3 with Aat2 and Pst1 and pSB1A10 with Nsi1 and Aat2 and ligated those fragments of each plasmid that contained our Bricks to get a Monsterplasmid.
We had to do so, as both plasmids contain the same ori mechanism. In addition, having both the switch and the signal RNA transcribed from the same plasmid gives us a high local concentration of the signal, once its transcription is induced.

We transformed BL21(DE3) cells with the plasmid. We set up cultures, induced the arabinose promoter and measured the GFP and mRFP1 excitation/emission spectra within time.

Unfortunatly, we were ot able to detect any RFP signal, not even in the positive control with no switch in between the GFP/RFP sequences.

From these experiments, we concluded, that the mRNA of the RFP variant used was instable and rapidly degraded by RNases, so the RFP was not synthesized in the cells. This was also the conclusion from XXX...

As solution to this, we decided to design a measurement plasmid ourselves:

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Second Try: A measurement plasmid of our own design

To design our own plasmid to overcome the problems that occurred in our first try gave us tghe possibility to overcome several other problems:

Third Try: One promoter for each protein

We decided to use the measuremnt plasmid we developed in our second try but to clone another L-arabinose induced promoter into the plasmid, in front of our switch followed by the mCherry sequence.

In this way, we still can use GFP fluorescence as internal control, because both protein transcription is under the control of a promoter of identical design.

Though we are still not able to tell exactly why our previous measurements did not work, but with this construct we measured the first time fluorescence of the mCherry protein in our positive control.

Discussion

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References

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