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.

Fig.2 Characterization of pT181 attenuator in DH5-α E.coli cells. The figures show the fluorescence for cells with or without pT181 antisense. (a) Fluorescence monitored over time for cell lines incorporating the pT181 system with pT181 antisense. It shows that the GFP can be expressed in the pT181-attenuator, and the expression level increases gradually. (b) Fluorescence monitored over time for cell lines incorporating the pT181 system without pT181 antisense. It matches the curve of how GFP’s expression increases without being repressed, which establishes foundation for measure the repression effect of pT181-attenuator. (c) The combination of (a) and (b). We could see the sharp difference in the fluorescence between the two curves. This proves our pT181 could repress the expression of GFP as expected, which means our part C is able to produces repression effect as anticipated. This shows that the controller in our Three-Node Feedback Loop is constructed successfully.

Fig.3 Characterization of pT181 attenuator in DH5-α E.coli cells. endpoint fluorescence (18 hours) for cell lines in the absence or presence of Pt181. The data shows that our Pt181 attenuator could repress the target gene for 98%.

Comparasion

Improvements compared with Kyoto pT181:
iGEM13_Kyoto:
Kyoto pT181: BBa_K1126003 http://parts.igem.org/Part:BBa_K1126003
We are utilizing pT181 attenuator – a dual control repressors, rather than only repressing on the transcriptional level – to regulate both gene transcription and translation in a fast and robust way.

In order to know how the characteristics change, we constructed a plasmid with both dual control pT181 antisense, dual control pT181 sense target and a GFP, as well as a plasmid with a dual control pT181 sense target and a GFP. We also construct a plasmid, in which the GFP is under the control of the pT181 antisense and pT181 sense target used by Kyoto.

Fig.4 A schematic representation of the experimental group plasmid. This has the basic pT181 attenuator Antisense under control of a constitutive promoter, as well as a GFP gene downstream of the pT181 attenuator sense target under the control of a constitutive promoter.

Fig.5 A schematic representation of the positive control plasmid with the GFP gene downstream of the pT181 attenuator sense target under the control of a constitutive promoter, without a pT181 attenuator Antisense on it.

Under the same cultivation environment and using same promoters and RBS, the expression efficiency of our dual control optimized pT181 is extremely higher than that of the Kyoto pT181 by picking some InterLab plasmids with different intensity as control groups.

Fig.6 Characterization of dual control pT181 system in DH5α strain with pSB1C3 as a vector. Fluorescence over time for cell growth incorporating dual control pT181 or Kyoto pT181 system. From the figure, we can see the fluorescence of the dual control pT181 is always lower than the Kyoto pT181, which means it shows better repression effect.

Fig.7 Characterization of pT181 attenuator in DH5-α E.coli cells. Endpoint fluorescence (18 hours) for cell lines under the control of dual control or Kyoto Pt181. The data shows that our Pt181 attenuator could repress the target gene for 15% than the Kyoto pT181.