Difference between revisions of "Part:BBa K1493501"

Revision as of 23:54, 11 October 2022

pRha + GFP

GFP controlled under an L-rhamnose inducible promoter (pRha).

This BioBrick is used by [http://2014.igem.org/Team:Wageningen_UR BananaGuard] as a control in the [http://2014.igem.org/Team:Wageningen_UR/project/characterization rhamnose mediated characterization].

Usage and Biology

Figure 1. Average GFP fluorescence of Escherichia. coli DH5α carrying a pSB3K3 plasmid containing the pRha gfp BioBrick. At t=0 the cells were induced with different concentrations of rhamnose in triplo. Fluorescence is measured in a plate reader in time (A) and at time point 8.25 (B) normalized for OD600.

The measurements in Figure 1 indicate a tuneable activation of pRha by rhamnose. The RFU values of 0% and 0.001% rhamnose are not significant taking into account the high standard deviation for these measurements as can be seen in Figure 1B. From a rhamnose concentration of 0.01% to 0.2% a significant increase in fluorescence is measured. Fluorescence from Figure 2 can be used to predict the concentration of repressor protein produced. Data points for time 8.25 h were chosen for the graph in Figure 2B due to the peak at time for 0.2% rhamnose, which is visible in Figure 1. This peak can be explained by the rhamnose depletion, as it is consumed by Escherichia. coli, causing the stop of promoter induction.

Team: BNDS_China 2021, improvements for biosensor

In our project, strains with higher rhamnolipid yield have to be selected. To achieve this goal, rhamnosidase is used to hydrolysis rhamnolipid to rhamnose and lipid, and the rhamnose yield could reflect the rhamnolipid yield of the strain.

Usage in project

GFP is being replaced with ORF of AmpR by Gibson assembly. This allows bacteria with more rhamnose to produce more ampicillin-resistant proteins. With more antibiotic resistance produced under the environment of ampicillin, bacteria that have higher in-cell rhamnose concentrations survive better. Subsequently, cells that can produce more rhamnolipids survive better since they can resist the harsh antibiotic environment. Therefore, this part helps us to automatically select desired mutants.

Quantitative Characterization

The following graph demonstrates the validity of this part. With higher rhamnose concentration, the concentration of the bacteria fluid tends to be greater, indicated by a high absorbance at OD600. The absorbance of culture with 0/20/40/60/80ul rhamnose added is measured every hour after liquid inoculation.

""

For further information, see the improved part: BBa_K3745000

Team: BNDS_China 2022, Contribution

Sequence and Features