Part:BBa_K3192012

T7A1 Promoter

The introduction of two plasmids bears a great metabolic cost to a cell. To decrease the energy wasted on translation in various parts of experimentation, the 2019 Virginia iGEM team the T7A1 promoter to regulate expression of two composite parts. This promoter is a tightly regulated, inducible, promoter that requires the presence of IPTG in the cell for translation to be initiated. Without the presence of IPTG, translation will not occur, but as soon as IPTG is added, transcription and translation ensue at a high rate.

Most all existing and well characterized T7 promoters require T7 polymerase for the tightly inducible transcription of a device. This T7A1 promoter does not require T7 polymerase for transcription, regular polymerase is sufficient.

This T7 promoter was characterized by Sclavi et al in 2004. These experiments were carried out studying this promoter in E. coli, a common iGEM chassis organism. This promoter is cited at “one of the strongest known prokaryotic promoters.” The consensus promoter on this sequence (TTGACT) has been shown to increase the rate of promoter binding and stimulate isomerization to the open complex.

Sclavi et al visualized the conformational changes that are undergone during promoter recognition and open complex formation by polymerase.

“RNA polymerase subunits β and β′ are in two different shades of green, whereas the α N-terminal domains are in light and dark blue. The α-CTD, absent in the crystal structure, have been added as blue spheres. The σ subunit is in red. A simplified cartoon of the DNA is drawn to scale. The TS is in dark purple and the NTS is light purple. The contacts shown between the enzyme and the DNA reflect the decreased solvent accessibility of nucleotides in specific positions on the promoter during the formation of the complex as determined in this work by time-resolved hydroxyl radical footprinting.” (Sclavi et al, 2008)

Testing the Functionality of BBa_K3192012

To test the functionality of BBa_K3192012, the 2019 Virginia iGEM team used composite part BBa_K3192031. This composite part encoded the necessary genes to synthesize polyhydroxybutyrate (PHB), a biodegradable plastic. The presence of PHBs in a cell culture can be determined using a procedure called Red Nile Staining. To determine if the promoter functioned properly, cultures of DH5-alpha E. coli expressing BBa_K3192031 were stained with Red Nile to compare PHB presence.

Pictured below is the control (BBa_K3192031 lacking IPTG) on the left, and BBa_K3192031 present with IPTG to test expression.

Red Nile staining of E. coli K12 expressing BBa_K3192031 which produces PHBs.

E. coli K12 cells expressing no genes for PHB production.

The control lacking PHBs indicates that IPTG is required for transcription to be initiated, confirming the tight inducibility of the T7A1 promoter without the necessity for T7 polymerase.

References:

1) Sclavi, B., Zaychikov, E., Rogozina, A., Walther, F., Buckle, M., & Heumann, H. (2005, March 29). Real-time characterization of intermediates in the pathway to open complex formation by Escherichia coli RNA polymerase at the T7A1 promoter. Retrieved from https://www.pnas.org/content/102/13/4706.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 1
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]