Part:BBa_K1126003

pT181 attenuator

pT181 attenuator is the locus where regulates the transcription of downstream gene with pT181 antisense (BBa_K1126006) if it is located between promoter and ribosome binding site.
In the absence of pT181 antisense, secondary structure of pT181 attenuator locus does not affect transcription. However, when mRNA of this locus is partly coupled complementary with pT181 antisense RNA, this locus is folded into Rho-independent terminator and terminates the transcription right before the gene. The efficiency of this termination is very high and downstream gene is strongly silenced (the efficiency is shown to be 84% in Escherichia coli).
This mechanism is originally found in the regulation system of plasmid pT181 in gram negative bacteria Staphylococcus aureus.

https://parts.igem.org/Part:BBa_K2787030

New Design

iGEM18_ShanghaiTech:
Dual Control pT181:BBa_K2787030 https://parts.igem.org/Part:BBa_K2787030
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]