Part:BBa_K2787030

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_ to 98%. The part achieve 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

A schematic representation of the pT181 attenuator in action

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.

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.

400x image of positive control under fluorescence microscope.

400x image of experimental group under fluorescence microscope

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.

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.

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.

The combination of the two figures above. 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 produce repression effect as anticipated. This shows that the controller in our Three-Node Feedback Loop is constructed successfully.

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%.


Improvements compared with Kyoto pT181:
iGEM13_Kyoto:
Kyoto pT181:BBa_K1126003 https://parts.igem.org/Part:BBa_K1126003
We are utilizing pT181 attenuator – a dual control repressors – 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.1

Figure 1: 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.2

Figure 2: 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 extreamly higher than that of the Kyoto pT181 by picking some Interlab plasmids with different intensity as control groups.

Fig.3

Figure 3: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.4

Figure 4: 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.5

Figure 5: 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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]