Measurement

Part:BBa_K398331

Designed by: Kira Schipper   Group: iGEM10_TU_Delft   (2010-10-07)

pCaiF-B0032 measurement device

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.


The characterization and results of BBa_K398331 are described on the BBa_K398326 (promoter) BioBrick page and on the TU Delft iGEM Team 2010 wiki



Team SEU-Nanjing-China's 2019 Characterization

Team SEU-Nanjing-China measured this part BBa_K398331.Our aim is to characterize the pCaiF promoter. We are very interested in this promoter. According to the description of this part, pCaiF is a natural promoter found in E. coli K12 , which regulates the expression of genes involved in the degradation of non-glucose carbon sources. The promoter is regulated by the level of cAMP. Because decomposition product of glucose auses decrease of cAMP, on the contrary, at low glucose level, the level of cAMP increases. CRP binds to cAMP to form complex cAMP-CRP. cAMP-CRP binds to pCaiF and activates the transcription of downstream components. Therefore, we hope to observe the experimental results: the expression of GFP in high glucose should be lower than that in low glucose.

1、We prepared LB medium with 5 different concentration of glucose, each concentration containing triplicate. The 5 concentration of glucose are 0.5g/L, 1g/L, 2g/L, 5g/L, 10g/L, LB medium without glucose as control. The E.coli grew in 15mL medium.

Figure 1. Single colony successfully expressing GFP.
Figure 2. LB medium with 5 different concentration of glucose, each concentration containing triplicate. The 5 concentration of glucose are 0.5g/L, 1g/L, 2g/L, 5g/L, 10g/L, LB medium without glucose as control.

2、Incubate at 37℃, 120rpm. We monitored the growth of cells by measuring OD600 every 1h.

3、After 10h, the OD600 reached 0.3-0.6. We measured GFP fluorescence and accurate OD600.

Table 1.We measured GFP fluorescence and accurate OD600 after the OD600 reached 0.3-0.6,and organize the data into this table.
Table 2.We measured GFP fluorescence and accurate OD600 after the OD600 reached 0.3-0.6,and organize the data into this table.

4、We construct Particle Standard Curve and Fluorescence Standard Curve, and use them to convert OD600 to cells amounts, and GFP fluorescence to the number of GFP molecules.

Figure 3.This is the cell concentration corresponding to the fluorescence intensity at different glucose concentrations.


Apparently, when adding 5g/L and 10g/L glucose, the amounts of GFP(per cell) is extremely lower than lower concentration of glucose. One interesting thing is that GFP(per cell) of group 1 and group 2 is slightly higher compared to the control. We guess that low concentration of glucose might play the role of nutrients and when there are more glucose, they can repressed GFP expression. Or there may be another mechanism we haven’t understand.

Figure 4.We construct the Particle Standard Curve and use it to convert OD600 to cells amounts. The right line chart is in the log scale and the left is in normal.
Figure 5.We construct Fluorescence Standard Curve and use it to convert OD600 to GFP fluorescence to the number of GFP molecules.The right line chart is in the log scale and the left is in normal.


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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 722


Functional Parameters: Austin_UTexas

BBa_K398331 parameters

Burden Imposed by this Part:

Burden Value: -0.6 ± 2.5%

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 BioBrick did not exhibit a burden that was significantly greater than zero (i.e., it appears to have little to no impact on growth). Therefore, users can depend on this part to remain stable for many bacterial cell divisions and in large culture volumes. Refer to any one of the BBa_K3174002 - BBa_K3174007 pages for more information on the methods, an explanation of the sources of burden, 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.

[edit]
Categories
Parameters
None