Part:BBa_K398326

Promoter of the CaiF protein

pCaiF is a promoter region from E.coli containing the binding site for the global regulator Crp which is expressed during periods of starvation. Under, for example, glucose limiting circumstances pCaiF activates transcription. pCaiF is tightly regulated by cAMP levels.

Introduction

pCaiF is a natural promoter found in E. coli K12, pCaiF is part of the translational unit of the protein CaiF which is a transcriptional regulator of carnitine metabolism under anaerobiosis and glucose limitation. From the original sequence, we took the cAMP-Crp and RNApol sigma 70 binding sites. Under low glucose levels, the cellular cAMP (cyclic Adenosine Mononucleotide Phosphate) levels are high and thus Crp (Catabolite gene activator protein) will bind easier to this molecule forming the complex cAMP-Crp. This complex acts as a transcriptional regulator of over 180 proteins involved in metabolism of secondary carbon sources.

Figure 1: Illustration of the mechanism of promoter pCaiF. At the left the promoter is off because there is enough substrate (glucose). At the right the promoter is on, activated by the presence of cAMP/crp

The original purpose of pCaiF for [http://2010.igem.org/Team:TU_Delft/Project/sensing TU Delft 2010 iGEM project] was to serve as a promotor for our proteins when glucose or other carbon sources easier to degrade are not in the medium, in that way cells will grow faster and once a certain cell density is reached they can start to degrade other carbon sources like alkanes.

Because of the limited amount of time, we just succeeded on the construction of a measurement device in order to characterize pCaiF. We hope that other teams will use these parts in the future for several reasons, among them the necessity of well characterized promoters in the catalog.

Characterization

The pCaiF promoter was characterized using GFP generator E0040. To determine the response in E. coli, an experiment in which different glucose concentrations were tested was performed. This was done in a 96-well plate and GFP and OD was measured online of the following strains: E. coli 331D (which contains BBa_K398331) and our negative control ( E. coli with J13002 in the plasmid pSB1A2). The protocol is explained in more detail on the [http://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=pCaiF_characterization_experiment TU Delft protocol page].

Results

The experiments showed a significant increase in GFP production when the initial glucose concentration was 2 g/L compared to the result for an initial glucose concentration of 10 g/L; whereas at 5 g/L a slight increase in GFP production at the end of our measurements was found. From these plots it can be concluded that the part is sensitive to cAMP levels. Moreover, it was found that the part is active under starvation periods (stationary phase at low glucose concentrations). The different plots were overlapped in order to show the differences between the various conditions tested.

GFP profiles in M9 medium at different initial glucose concentrations.

This system has also been modeled, see the [http://2010.igem.org/Team:TU_Delft/Modeling/pcaif-model TU Delft 2010 pCaiF model] More details on the [http://2010.igem.org/Team:TU_Delft/Project/sensing TU Delft 2010 iGEM wiki].

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]

Functional Parameters: Austin_UTexas

Burden Imposed by this Part:

Burden Value: 5.3 ± 2.4%

Burden is the percent reduction in the growth rate of E. coli cells transformed with a plasmid containing this BioBrick (± values are 95% confidence limits). This part exhibited a significant burden. Users should be aware that BioBricks with a burden of >20-30% may be susceptible to mutating to become less functional or nonfunctional as an evolutionary consequence of this fitness cost. This risk increases as they used for more bacterial cell divisions or in larger cultures. Users should be especially careful when combining multiple burdensome parts, as plasmids with a total burden of >40% are expected to mutate so quickly that they become unclonable. Refer to any one of the BBa_K3174002 - BBa_K3174007 pages for more information on the methods and other conclusions from a large-scale measurement project conducted by the 2019 Austin_UTexas team.

This functional parameter was added by the 2020 Austin_UTexas team.