Ecoli.PROM: an Erasable and Programmable Genetic Memory in E. coli

Figure 1: Genetic circuit

The specific goal of our project was to design a bacterial reprogrammable memory, i.e. colonies of genetically engineered E. coli immobilized in solid medium where they work as an array of binary memory cells.

To engineer the bacteria we designed a modular genetic Flip-Flop composed of two parts (Figure 1): a binary memory block (toggle switch) and an induction block sensitive to UV radiation to set ON the memory. UV has been chosen to have a fine spatial selectivity in programming the memory cells, whereas IPTG should be used to reset the entire memory. The fine tuning of the circuit elements was achieved by a model-based analisys.

The core elements of the genetic memory are two mutually regulated promoters, each designed as indipendent operator sites flanking a constitutive promoter. In this way, the promoter transcriptional strength and the repressor binding affinity can be independently fixed. To this aim we designed operator libraries for LacI, TetR, Lambda and LexA repressor cloning them in the BioBrick format for their standard assembly. This will allow the rational design of regulated promoter elements that are still lacking in the Registry.

This approach has been applied in the building of our UV-programmable memory, and we expect it to be a general benefit in a larger number of applications in Synthetic Biology.

Operator site library standardization

Even tough transcriptional regulation still plays a central role in synthetic biology, a modular assembly of regulated promoters and characterization of their properties has not been formalized, yet. Even in the Registry, each promoter, even if complex, is treated as a “standalone” monolithic element. At present state, the choice of a regulated promoter implies a prefixed sigmoid regulation curve. The only option is to choose a discretional scaling factor in the transfer of the transcription function to the protein through an appropriate RBS. Moreover, the choice of one specific transcription factor limits the choice to one or few possible promoter. The assembly of regulated promoters as the combination of such modular parts, as transcription factor binding sites and operators, could permit the rapid design of devices with desired regulation curve. In fact, in this way, promoter transcriptional strength and repressor binding affinity could be independently fixed.

A first step in the direction of promoting elements rationalization has been done the past year with the inclusion in the Registry of a family of constitutive promoters (Thanks to John Anderson---- mettere il link al Registry). Each element differs from the other members in the family just for few base pairs in -35 and -10 regions, keeping constant the rest of the sequence and giving rise to a different level of transcription spanning. We decided to use this valuable work as a platform, a starting point for a deeper and more general design strategy.

To pursue this aim, we designed an operator sequence library for four commonly used repressor proteins: LacI, TetR, cI and LexA (link figura). For each of these repressors, we got three sequences from the literature (link) with different repressor binding affinities (link Registry), to get a fine modulation of promoter sensitivity to repressor(see Table 1). Then, we isolated each single operator from the library (link al metodo) to assemble it into standard plasmids from the Registry of standard parts.

Once we get single operators or a combination of them, we can decide to assemble them in different position relatevely to promoter -35 and -10 sequences. It is known from the literature (Cox et al) that the position of an operator site, respectevely to the promoter, plays a crucial role in determining repression or activation. Moreover, even different repression levels can depend on the operator position. The three possible "locations" for operator sequences are:

- the distal region - upstream the -35 sequence - the core region - between the -35 and the -10 - the proximal region- downstream the -10 sequence

Since genomic position affects the operator effect on promoter activation, we decided to take the Berkley's costitutive promoter library as a good "collection" from which we could choose the ideal promoter, depending on the desired transcriptional strengh. Chosen the promoter, we planned to change the operator position to study the effect of position in the design of specific promoter with a derired behaviour.

At the first, we designed a synthetic circuit, composed of the Lac operator sites downstream of the BBa_J23118 promoter controlling the LacI and GFP protein synthesis. These constructs, were meant to allow the characterization of the operator- repressor binding affinity effect on promoter activation.

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Flip-Flop logic circuit

Figure 2: JK Flip-Flop

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Collaboration with other iGEM Teams 2008

• After the last year competition, at the beginning of the 2008, we decided to get a new team started for iGEM2008 competition. In April, we got in contact with Prof. Paolo Magni, who wanted to start a new team in Pavia for iGEM2008. So, in order to share experiences and ideas about iGEM, and to show him what kind of wet lab resources are necessary to develop a Synthetic Biology project, we met at the Cellular and Molecular Engineering Laboratory of the University of Bologna- Cesena Campus. After this first meeting, there have been other chances to meet during the summer. In particular, several conference calls were organized and two meetings were scheduled in Pisa and Bressanone (Italy). It was fundamental to compare lab protocols and techniques to help each other avoiding mistakes and speeding up project progress. The main topics of our discussion were the optimization of plasmid resuspension and ligation reaction steps as well as how to measure fluorescence. Finally, before DNA Repository quality control publication on the Registry web site, we cross-checked some parts that showed problems after DNA transformation. Problems had been confirmed by quality control results (parts' sequences classified as "inconsistent").
• We want even to mention the courtesy of the Valencia iGEM Team, that have advised us about the critical use of GFP and RFP at the same time.

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Project Details

In this paragraph you can find some information about main project topics.

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Results

Concluding the iGEM 2007 Project

In the iGEM 2007 we used the LacY gene (BBa_J2210) to design the genetic Schmitt Trigger. Since this part was not working well, we sent it to be sequenced and found that it contained a 35 bp insertion upstream the endogenous LacY gene sequence. This insertion probably caused a frame- shift in protein translation, making the gene ineffective. So, we amplified the right gene sequence and put it in the BioBrick format. Successive sequencing confirmed the right assembling of this part. We also measured IPTG-induced fluorescence in the genetic Schmitt Trigger (see Figure) and we assessed the correct function of the new LacY part. To contribute to registry’s improvement we decided to send this new part to the Registry ([http://partsregistry.org/Part:BBa_K079015 K0790015]).

Schematic representation of the genetic Schmitt Trigger. The LacI generator module was included to have a constitutive synthesis of LacI repressor protein witch makes up for endogenous LacI. LacY permease introduces a positive feedback. GFP is the reporter for pLac activation. This circuit express high level of fluorescence with very low concentration of inducer (IPTG=1uM). After switching, the fluorescence level f is insensitive to the increase of inducer dose.