Difference between revisions of "Part:BBa K1493501"

Revision as of 02:28, 14 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.

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For further information, see the improved part: BBa_K3745000

Team: BNDS_China 2022, Contribution

Introduction

Last year, our team (BNDS_China 2021) used the rhamnose induction system to induce antibiotic resistance genes as part of the directed evolution system. Although team iGEM14_Wageningen_UR had characterized the rhamnose operon previously, Data about its dynamic range, optimal inducer concentration, and leakage are still insufficient. This year, part of our project focused on the efficiency of induction systems. Thus we plan to fill in the lacking data of the rhamnose operator using kinetics. By finding the steady state (the proteins' generation and degradation at the same velocity) of the rhamnose-induced protein expression, we are able to directly relate the protein expression level to the concentration of rhamnose.

Quantitative Charicterization

We build a rhamnose-induced GFP expression plasmid for characterization. The steady state could be found by looking for a stage that GFP fluorescence/ABS 600 is stable. 8 Groups of different RHA concentrations: 0mM, 0.1mM, 0.5mM, 1mM, 2.5mM, 5mM, 7.5mM, 10mM, 20mM each with 6 repetitions were tested with proper controls (blank, WT strain, and constitutive GFP expression strain) in the same assay. ABS 600 and GFP fluorescence were read every 10 minutes, and the total duration of the program was 24 hours.

Fig.1: The layout of the kinetics assay

By finding the steady state of strains in each group of concentration, we build a standard curve (fig.1) of different concentrations of RHA induction. With higher rhamnose concentration, the ABS 600 of the bacteria culture will be higher when reaching a steady state. The critical point of rhamnose's concentration is 10mM. At 10 mM, the GFP fluorescence/ABS 600 in the steady state reaches the maxim value; after 10mM, there is no significant growth in GFP fluorescence/ABS 600 as the concentration of Rha increases.

Fig.2: The value of fluorescent level/ABS600 in different concentrations of rhamnose in their steady state.

Sequence and Features