Team:Bologna/Project

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The logic system consists of a toggle switch specifically modified to optimise and ease the reaching of the two levels and strengthen their stability over time. Additionally, the apparent redundance within the circuitry would allow higher level of modularity. For instance, it is possible to maintain unaltered the fundamental "brick" via feedback provided by the above mentioned toggle switch; this would allow substitution of the lo Po couple with another compatible module of interest. Another useful and funny application would also be the generation of images via fluorescent emissions from the matrix itself. In fact it would be possible to finely tune the frequency of the light representing each pixel, de facto chaging also the resulting colour of such pixel within the 2D picture in output.
The logic system consists of a toggle switch specifically modified to optimise and ease the reaching of the two levels and strengthen their stability over time. Additionally, the apparent redundance within the circuitry would allow higher level of modularity. For instance, it is possible to maintain unaltered the fundamental "brick" via feedback provided by the above mentioned toggle switch; this would allow substitution of the lo Po couple with another compatible module of interest. Another useful and funny application would also be the generation of images via fluorescent emissions from the matrix itself. In fact it would be possible to finely tune the frequency of the light representing each pixel, de facto chaging also the resulting colour of such pixel within the 2D picture in output.
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= Conclusions =
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This will allow the rational design of regulated promoter elements that are still lacking in the Registry.
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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.
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[https://2008.igem.org/Team:Bologna/Project ''Up'']
[https://2008.igem.org/Team:Bologna/Project ''Up'']
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= Conclusions =
 
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This will allow the rational design of regulated promoter elements that are still lacking in the Registry.
 
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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.
 
= Concluding the iGEM 2007 Project =
= Concluding the iGEM 2007 Project =

Revision as of 17:54, 28 October 2008

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HOME PROJECT TEAM SOFTWARE MODELING WET LAB LAB-BOOK SUBMITTED PARTS BIOSAFETY AND PROTOCOLS


Contents

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 and an induction block sensitive to UV radiation to set LacI ON. 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 (TetR ON).

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 allowed us to design and assemble three different circuits, where the BBa_J23118 constitutive promoter was cloned with the Lac operator 1, the Lac operator 2 and the symmetric one, from the Lac operator library. Promoter activation was repressed by the LacI repressor depending on the operator- repressor binding affinity. LacI was cloned downstream of the promoter- operator sequence and the GFP was chosen as the reporter. These circuits were planned for the determination of the Ki transcription index (link).

The fine tuning of the circuit elements was achieved by a model-based analisys.



Conclusions

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.


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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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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 (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.


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