Difference between revisions of "Part:BBa K808000"

Revision as of 22:18, 26 September 2012

araC-Pbad - Arabinose inducible regulatory promoter/repressor unit

This part contains the promoter as well as the coding sequence for the repressor AraC which is transcribed in the opposite direction. (“upstream”) By binding to L(+)-arabinose, AraC changes its conformation. This causes the protein to diffuses from the DNA thereby inducing transcription.

Usage and Biology

• Inducer: L(+)-arabinose
• L(+) - Arabinose is a sugar and ist harmless. For overexpression of proteins the concentration of 0,001-0,02% Arabinose can be used.
• We designed this biobrick in order to get a promoter with low background activity so that our expressed membrane proteins wouldn’t damage the cells before induction.
• We also needed an adjustable regulation of induction so that we could induce different levels of transcription, as membrane proteins might damage the cells when expressed at high levels according to the small capacity of enrichment in the membrane.

Sequence and Features

Charakterization

Charakterization using PET cleaving enzyme pNB13

This biobrick did measure up to our expectations as shown in the following data. We also used this promoter to express our PET cleaving enzyme pNB-Est13, which is anchored C-terminal to EstA.(E. Coli membrane anchor protein). AraC-Pbad shows a low background activity and a good respose to the induction with Arabinose. For more details: BBa_K808032

Figure 1. Cells grown in GFP-medium containing the arabinose promoter regulating GFP expression. Induced at time=0h.

Charakterization using GFP

The promoter was characterized using GFP to measure gene expression at different arabinose concentrations. Cells were grown in 24-well plates in a defined medium ([http://2012.igem.org/Team:TU_Darmstadt/Materials/GFP-Medium GFP-medium)] that provided fast growth without interfering with fluorescence measurement. Measuring in LB-medium turned out not to be possible.

Results Cells showed fluorescence after 4-5 hours when grown in a medium containing >0.01 % (w/v) of arabinose. The response of the promoter to different concentrations turned out to be dynamic, and different levels of gene expression were inducible. E. coli DH5alpha was used for the measurements.

Figure 2. Cells grown in LB-medium containing the arabinose promoter regulating GFP expression. Induced at time=0h.The culture with 0 % Arabinose shows no GFP expression, the cultures with 0,3 % Arabinose show a response after about 1 h and the culture with 0,5 % Arabinose shows a response after a short time.

GFP response in LB-medium

For overexpression of our genes we wanted to grow the cells in LB-medium. To find out how we had to scale and time gene expression we made another test using GFP. Because of the high background fluorescence it was required to transfer cells in H2O before measurement.

Results The response was larger in LB-medium compared to GFP-medium. Reasons for this might be the faster metabolism as well as slight presence of arabinose in LB-medium. This could lead to higher expression of receptors targeting arabinose and a faster response. Measurements were done in E. coli DH5alpha

Reference

• Schleif, R. (2000). "Regulation of the L-arabinose operon of Escherichia coli." Trends Genet 16(12): 559-565.
• Ren, H., D. Yu, et al. (2009). "High-level production, solubilization and purification of synthetic human GPCR chemokine receptors CCR5, CCR3, CXCR4 and CX3CR1
• http://openwetware.org/wiki/Titratable_control_of_pBAD_and_lac_promoters_in_individual_E._coli_cells#pBAD_promotersOpenWetWare
• http://www.ncbi.nlm.nih.gov/pubmed/7768852?dopt=Abstract