Difference between revisions of "Team:ShanghaiTech/Improve"

Improved

pT181 Attenuator target sequence with improved repressive efficiency

An improved version of pT181 sense target with its length shortened from 287bp to 201bp and its repressive efficiency improved from 84%for BBa_K1126003(http://parts.igem.org/Part:BBa_K1126003) to 98%. The part achieves such efficiency through forming secondary structures that can interact with the pT181 antisense and hinder the transcription of downstream genes. With the absence of pT181 antisense, nevertheless, the expression is basically unaffected.

Usage and Biology

Our engineered cells need a Three-Node Negative Feedback Loop to construct a more sensitive and high-fidelity control system. And pT181 attenuator is the part that plays the role of repressor in this loop.
pT181 attenuator is a part composed of a sense target sequence and an antisense RNA that can regulate gene transcription and translation. Residing in the 5΄ untranslated region of the target gene, it can regulate the expression of a downstream gene at both transcriptional and translational levels.
The RNA regulators show sufficient advantages over traditional protein-based regulatory systems, including:
-- Programmability: As Watson-Crick base pairing is predictable, the RNA-RNA interaction can be predicted by sophisticated software tools. In this way, a RNA switch can be designed artificially, which are difficult for proteins.
-- Lower metabolic cost: Compared with proteins, the RNA switches dispense with translation step, which saves a great amount of resources.
-- Fast response: RNA switches could propagate signals faster than proteins considering the fast degradation rates of RNAs.

Despite these advantages, RNA regulators still suffer from incomplete repression in their OFF state, making the dynamic range less than that of the proteins. This leak can cause the network to function incorrectly. Therefore, we submit the dual-control pT181 attenuator, which can solve this problem.

The dual-control pT181 attenuator we submitted offers a significant advantage over previous iGEM parts that submitted in 2013:
-- Reduce leak: As our pT181 attenuator could regulate both transcription and translation in a single compact RNA mechanism, which means it could provide stronger functions without increasing burden. This dual control repressor is able to increases repression from 85% to 98%.

Characterization

We generated two plasmids, one is for experimental group, the other is for a positive control. The experimental plasmid, which is the pT181 attenuator in this experience, contains the antisense sequence downstream of a constitutive promoter and followed by a double terminator on a high-copy plasmid. Meanwhile, there are also a GFP gene with a ribosome binding site downstream of the pT181 attenuator sense target sequence. The GFP coding sequence is also downstream of a constitutive promoter and followed by a double terminator. The positive control plasmid, which is the blank in this experience, contains the same as the experimental plasmid except for the antisense sequence.
We did a group of pT181 attenuator expression experiments. First, as a part that needs to show strong inhibition, we should ensure that its inhibitory effect is obvious enough. Therefore, we compared experimental group and positive control group, which is transformed into a normal GFP plasmid. Depending on the GFP expression, we can prove that our work has a high credibility. Additionally, in the group above, three types of flora from InterLab are cultivated for contrast. The aim is to compare the statistics of pT181 attenuator and verified InterLab to find out which repressor level pT181 attenuator is in when put into practical application.
For this group, we transform different plasmids into the E. coli in the tubes and cultivate for hours (37℃, 220RPM). Then we used ELISA plate to detect the change of fluorescence and OD600 over time. What should be noticed is that we set the original flora at OD600=0.05 to guarantee flora proliferating at the same concentration. As the repression of pT181 attenuator attenuator is so powerful that the fluorescence of the experimental group is hard to detect. As a result, to remove LB medium’s fluorescence background, we centrifuge fluid, take out supernatant, add PBS buffer and resuspend before detect.

Fig.1 Characterization of pT181 attenuator in DH5-α E.coli cells. OD600 monitored over time for cell lines incorporating the pT181 attenuator in the absence or presence of the pT181 antisense. The result shows that the pT181 antisense is not harmful to the E.coli, which provides convenience for test for fluorescence as we do not need to normalize the OD600.