In principle we decided to use an expression cassette consisting of Green Fluorescent Protein (GFP) coding sequence upstream of the switch and another fluorescent protein coding sequence downstream of it. Both protein coding seuqence carry the same ribosome binding site, therefore, the GFP fluorescence can be used as internal control in measurements. Since the spectra should not overlap and to avoid FRET as well as an pure overlap of the spectra, we settled on the usage of red fluorescent protein variants, namely mRFP1 in the first try. While the GFP fluorescence is used to normalize the measurements, the RFP fluorescence is used to detect termination/antitermination.

Upon binding of the signal the stem loop of the switch would resolve leading to red fluorescent additional to the green one of GFP. The internal control carries the advantage that errors in the measurement set can be detected easily. Lack of arabinose or promotor insensitivity can be recognized as well as problems with the fluorescence measurement itself. Plus, we have a way to normalize our measurements and compare different preparations in relation to each other.

Our measuring plasmid is based on the BioBrick pSB1A10, A1, distribution 2010. Unfortunately after two months of cloning we had to recognize that the plasmid in use did not work (see also Biobrick validation--> link). So after the first unsuccessful attempts we had to reclone the system, substituing RFP to mCherry, a dsRED derivative with a spectra in the far red, and adding arabinose inducible promoters in front of both fluorescent proteins.

To control the expression of the switch, the particular DNA sequence itself is under the control of an IPTG dependent promotor. In the future we want our networks to be able to respond to a variety of external signals like small metabolites, ions or whatever can be found in the parts registry. For a start we went with an established and well-working system like the lac-operon. ---> PLASMID MAP
So upon induction with arabinose a rise of GFP expression can be seen. To monitor changes in gene expression we put our E. coli cells in a fluorimeter and measured fluorescent. It worked quite nicely with living cells in a fluorimeter, the only thing to avoid is too much scattering: the cell density should not exceed an OD600 of ???. Continious stirring and a set temparature at 37°C allowed measuring over severall hours. Cell density was checked in between.

For switch evaluation, IPTG was added to the cells after about two hours after arabinose induction (baseline). ??? Stimmt das?? A rise of RFP/mCherry emission should be visible in case of a working switch.
For evaluation of the measuring plasmid itself we incorporated a positive control in every measurement. A random sequence in between GFP and RFP/mCherry was chosen in a corresponding length. An increase in both GFP and mCherry was detectable in the positive control and in the same amount after quantum yield correction, proving that the measuring plasmid is working nicely.
As a negative control we measured the same plasmids as for every switch but without signal. Since our switches are effective terminators if no signal is bound, transcription can not occur and no RFP/mCherry is produced.

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:

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

So haben wir in vivo translation gemessen

Since our switch is RNA-based and the whole mechanism takes place on trancriptional level, we looked for a method to check for termination efficiency only on RNA-level. In vitro transcription offers an elegant way for a fast and easyn prove of principle. If measureable effects with our basic concept can be seen in vitro we can use the so gained data to optimize the system in vivo. Since we are working on a totally new principle of trancriptional control, we used this approach for easy variation of different variables like the length of the core unit and the switch to signal ratio. 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.
Working with in vitro systems also has the advantage that an input is not needed anymore and the output can also be generated easily. We used two readouts with two different transcription systems to check and investigate our devices: First, we used an malachitegreen-binding aptamer for an fluorescent output (which will be described in detail later) and second, we simply put our reaction educts on an denaturing acrylamide-gel to check for RNA varying in length. As for two different transcription systems we used on the one hand E. coli-RNA Polymerase (RPO) based transcription since the aim is to apply our system in vivo and on the other hand T7 based transcription which is well established through literature.

T7 RNA polymerase

The T7 RNA polymerase is known for satisfying RNA yields together with easy handling. In our approach we had PCR amplified, double stranded switches with an malachitegreen binding aptamer following after the switch (133 bp, see section below) and a single stranded signal with about 30 bp length.

For in vitro expression the T7 RNA Polymerase requires a double stranded promotor region at the beginning of the DNA template but is otherwise capable of handling single stranded DNA, so a sense strain corresponding to the T7 promoter region was added. Transcription is more effective with double stranded DNA as template. Since we ordered the signal sequences we tested we chose the cheaper way in the beginning by using single stranded signals with corresponding sense T7 pieces and switched to double stranded constructs after narrowing down the most promising switch/signal pairs.

E. coli RNA polymerase

Denaturing Polyacrylamide gel electrophoresis

We also used Polyacrylamide gel electrophoresis (PAGE) for evaluation of termination efficiency of our basic units. Gels containing 15 % acrylamide and 6 M urea were used for separation of 90 (terminated by switch) and 133 bp (continous reading) RNAs.

Polyacrylamide gels seperate RNA and DNA according to their size in an electrical field. Since the negative charge equals the size of nucelotides in the RNA/DNA, the number of base pairs can be compared between two samples. Since RNA forms threedimensional structures, the samples are preheated and run in 6 M urea. The polyacrylamide gel is stained in SybrGold afterwards which binds to both single and double stranded DNA and RNA.

Denaturing PAGE is a simple yet elegant way to check for transcription efficiency and termination rates.

Malachite green assay

Emission spectra of malachite green; A: without signal-RNA, B: with signal-RNA

Malachite-green is a dye with a negligible fluorescence in solution but undergoes a dramatic increase if bound by a RNA -aptamer. Upon binding to the aptamer, the fluorescence of malachite-green increases about 3000 times making it an exceptionel good marker. Since the binding is very specific, transcription in dependence of a signal can be monitored by measuring the fluorescence of malachite-green over time if the aptamer is located behind the switch. Transcription of the aptamer will only take place after anti-termination by a signal. An increase should be visible over time.

For the T7-based measurements we ordered single stranded signals for a first attempt and added matching single strands complementary to the T7 promoter region. The switch was amplified using PCR and consisted of the following elements: Primer-binding site - T7 promotor - switch - malachitegreen binding aptamer. Upon binding of a correct signal to the switch, the stem loop dissolves and transcription is possible.

OLD: 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. The RNA-aptamer

---concept to be described, as well as literature--- <ref>refs</ref>

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.

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