Part:BBa_K808000

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.
• Can with success be combined with a promoter from pBAD SPL to obtain a very low leakiness and an appropriate strength. This is very efficient for expressing lethal proteins.

Sequence and Features

Assembly Compatibility:

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

  Illegal BamHI site found at 1144
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961
  

Characterization

Characterization 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.

Characterization 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.

Characterization using GFP in S30 cell-free extract

Methods:

For characterisation of the leakage of the promoter, we amplified our constructs containing BBa_k80800 in E.coli and performed plasmid minipreps to extract circular DNA for expression in a cell free system. We then sequenced our plasmid DNA.

For characterisation of BBa_K808000 in response to arabinose concentrations, we prepared a cell free system containing BBa_K808000 from the parts registry and left it to incubate for 10 hours at 37°C. The cell free system we used was the E.coli S30 extract system for circular DNA from Promega. We then added 1 μl of arabinose at the concentrations of: 0.05%, 0.1%, 0.5%, 1% and 2% and ran the experiment for 10 hours at 37°C, recording fluorescence intensity every 10 minutes.

Results:

Characterisation during amplification of our constructs:

Sequencing analysis showed that we successfully amplified the part BBa_K2206006 (this part contains BBa_K2206000, BBa_E0040 and BBa_K808000) with no fidelity errors. Therefore BBa_K808000 was suitable for preventing toxic levels of our part BBa_K2206006 in E.coli, demonstrating that BBa_K808000 has low levels of leakage. However, we found some mutagenesis in the promoter (BBa_K808000) for part BBa_K2206007 (which contains BBa_K2206001, BBa_E0040 and BBa_K808000), meaning toxic levels of BBa_K2206007 were still produced. This indicates that the promoter has some leakage and may therefore be unsuitable for regulating the expression of lethal parts.

Characterisation using GFP:

We found that fluorescence increase occurred in as little as one hour and maximum fluorescence was reached after ~8 hours for all the concentrations. We found that fluorescence increase occurred with as little as 0.05% arabinose (we did not measure lower than this) and increased with arabinose concentration up to 0.5% arabinose. Interestingly, we saw a decline in fluorescence intensity at 1% and 2% arabinose concentrations. We are unsure as to why this happened, but we speculate that the arabinose altered the pH of the cell free system, causing some protein denaturation.

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