A key aspect of this project is the conjugation system which allows the signal plasmid to be transmitted from cell to cell. A thorough design plan was drafted and we set about building our communication system in the lab. Performing the necessary knockouts proved to be a difficult task. Three different knockout attempts were made using the λ-red system without any success. The exact reason for this remains unclear. Although we were unable to successfully knockout the required genes from the R751 conjugative plasmid, progress was made with the signal plasmid. Despite the fact that we did not have access to R751 ΔoriTR cells, we were able to show that our prototype signal plasmid is transmitted in the presence of wild R751. This plasmid should provide a useful tool for future teams wishing to use the R751 system. Another success was the synthesis and characterization of the trbK entry exclusion protein. Conjugation tests showed that this was able to block incoming conjugative transfers, as predicted.
Self Destructive Plasmid
The goal of the Self Destructive Plasmid subproject was to create a plasmid that could be induced to destruct itself. It would do so by expressing an endonuclease that would recognise and cut a sequence in the same plasmid. This linearised DNA was expected to be automatically degraded by the cell.
The final goal of self destruction was not reached. However, a 30 basepair I-SceI homing endonuclease restriction site has been biobricked and shown to be restricted by that endonuclease. It has been combined with an inducible GFP-LVA reporter gene, which has also been shown to work.
The problem is the I-SceI homing endonuclease. First a part from the registry was used, that turned out to be T4 DNA ligase instead of I-SceI homing endonuclease. Construction of this part was done by assembling more basic parts. But the gene for the I-SceI enzyme turned out to have an LVA degradation tag attached to it. This shortens the life time of the protein inside the cell considerably and possibly prevents normal folding. It is hypothesised that disfunctional I-SceI is the reason that the expected Self Destruction did not occur. However, a contribution to the registry was made by documenting the anomalies in the biobricks containing the I-SceI homing endonuclease.
Synthetic Transcriptional Cascade
In nature, a fundamental “device” in gene regulation circuits is time-delay responses to internal or external stimuli. In the present work, one important feature performed for our system is the reset of the signal once the message is received. The time between when the signal (or self destructive plasmid/conjugation plasmid) has been received and the reset (or destruction of the plasmid) is an important parameter which will allow the signal to be sent to the next cell (receiver) through the consecutive conjugation system before “losing” the message. Therefore, it is necessary to construct a device which will give enough time for the two subsequent events happen. This device is termed as the time-delay genetic circuit in this project.
A functional time-delay device was designed, constructed and delivered to iGEM registry. This device is based on transcriptional regulation and has the capability to integrate an input signal, IPTG, and trigger the expression of GFP after a delay from the initial detection event. The device is formed by two new functional biobricks, this division not only allows the transfer of the helper plasmid in TUDelft 09 's project but also allows other teams to construct larger or shorter cascades which will deliver, after proper design, longer or shorter time-delay behaviors.
The plasmids constructed in this work were proved to work individually as expected and once they are transformed together within the same cell, they perform as a delay device. In the experiment performed in this project, the expression of GFP was substantially increased at 600 mins after induction with 0.2 mM IPTG.
From the observation K12 positive control strain used in experiment glowing GFP, makes us to hypothesize that the lack of enough LacI did not allow performing a better characterization of the device due to a lack of proper control. However, in relative measurements, the device was proved to work.
Biosynthetic AND gate
It was expected that post-transcriptional regulation, generated synthetically by the lock of the RBS and the following induction of the key for that lock will create a time-delay device. A plan for this strategy was elaborated in this sub-project and several attempts to construct it were performed. However, none of them were successful and this strategy was left behind in order to complete remaining work.
Lock and Key Library
Based on the work of Isaacs F. 2004, Berkeley iGEM 2006, Caltech iGEM 2007 and Peking iGEM 2007 and impulse by the lack of riboregulators, or named as locks and keys in iGEM members, we present the design and construction of an algorithm of an online lock and key generator. This work provides a tool for future iGEM teams which wanted to use post-transcriptional modification as a regulatory system and, because design needs, the use of RBS’s with different strengths are required. Although several teams have designed different riboregulators, they always focused on the “normal” RBS from the registry; this does not take advantage of the large library of RBS present in the site. Moreover, various teams make use of this post-transcriptional tool in this year projects (for example KULeuven and Tokyo-Nokogen), indicating a need for improvements and expansions of this tool.
The predicted secondary structure of the RNA derived from the DNA generated by the online generator, shows that the locks sequester the RBS chosen by the user, impeding the expression of the protein downstream the RBS. The online tool also generates a matching key which interacts with the lock will release the RBS, allowing expression of the protein of interest.
In order to test the functionality of the riboregulators generated by the online tool, two pairs of lock and key were chosen to be constructed and tested. The locks and the respective keys for “weak” and “medium” RBS from the registry were successfully constructed. These riboregulators were used to construct two plasmids which could help for the characterization of the locks and keys. Besides the mentioned plasmids, two controls were designed and constructed. Both, controls and plasmids, were constructed to be functional after IPTG induction. However, the lack of LacI repressor in the cells used forbids us to completely characterize the riboregulators. Future work should be done in order to characterize this new biobricks and other generated by the online tool created in this work and increase the tools for control of gene expression.
The undervalued but key ethical issues in synthetic biology mainly include reductionism and the bottom-up approach. Issues concerning safety, security and handling of intellectual property rights should mostly be related to politics not ethics. Instead there should be more focus on questions concerning life, specifically the reductionist (top-down) approach towards understanding life, and the bottom-up approach towards creating life. The issues are evaluated by a literature study and a survey based on that study, amongst 60 supervisors (and advisors) and 168 students involved iGEM 2009. The headlines:
- Participants believe that the value of life cannot be changed by synthetic biology
- Craig Venter's research is presumed not to involve creating new life forms, but rather applying genetic engineering
- Safety and security issues are acknowledged and additional regulation should minimize their risks
- Synthetic biologists are generally careful, but the public wants more
- There is no relation between the existence of a God and sacred or holy properties of life
- Communication with the general public is difficult but perceived necessary
- Life can possibly never be explained by mankind
- The reductionist approach towards understanding life is limiting