Team:Aix-Marseille/Contribution
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Contribution
Studied Part
- Name in Registry : BBa_J04500
- Group: Aix-Marseille 2021
- Author: Liam Zamora & Yassmine Amara
- Summary: Testing IPTG induction and glucose repression of BBa_J04500
IPTG induction ability measurement
An expression plasmid for sfGFP was constructed by cloning BBa_K3788013 (pelBsfGFP coding sequence) in BBa_J04500 (plasmid holding a supposed IPTG inducible promotor). It was transformed into DH5a E. coli competent cells. After transformation and overnight liquid cultures, green colonies were observed on LB+Agar plates containing IPTG (induced cells) or not (not induced). These results seemed surprising and this is the reason why IPTG induction was tested and quantified.
To test the ability of the promoter to be induced by IPTG, two types of liquid cultures were made : with bacteria containing BBa_J04500+BBa_K3788013 plasmids (plasmids cloned with the sfGFP coding sequence) or containing the empty palsmid BBa_J04500 (negative control). They were then incubated with different concentrations of IPTG for 3 hours (0 to 1 mM) as presented in Table 1. After induction, fluorescence intensity was measured for each condition at different OD600 (Figure 1).
| Condition | IPTG concentration (µM) | |||||
|---|---|---|---|---|---|---|
| BBa_J04500 + BBa_K3788013 | 0 | 10 | 20 | 50 | 100 | 1000 |
| BBa_J04500 | 0 | None | 1000 | |||
Figure 1: Fluorescence intensity obtained for each induction condition. Induced culture of cells containing the BBa_J04500 plasmid cloned with the sfGFP coding sequence studied at OD 0.02 (green), OD 0.2 (yellow) and OD 2 (orange); Induced culture of cells containing the empty BBa_J04500 plasmid studied at OD 0.02 (light blue), OD 0.2 (grey) and OD 2 (dark blue)
It seems adding IPTG to the growing medium does not affect the fluorescence intensity in bacteria transformed with the cloned vector, meaning it does not enhance transcription of the GFP encoding sequence. Moreover, as expected, very low fluorescence was measured with bacteria containing the empty vector (no GFP expression).
An hypothesis for theses could be that the cellular LacI quantity is not sufficient to repress the gene transcription ; or that the promoter is not responding to repression and therefore cannot be induced by IPTG.
Glucose repression ability measurement
As no difference in protein production was seen while using IPTG and given the previous hypothesis, glucose repression of the system was tested.
To test the ability of the promoter to be repressed by glucose, the two same types of liquid cultures as mentionned before were done (BBa_J04500 cloned with the sfGFP coding sequence or empty BBa_J04500). They were incubated with different concentrations of glucose (given in Table 2) and fluorescence intensity was measured every 60 minutes.
| Sample | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
|---|---|---|---|---|---|---|---|
| Glucose content ratio (v/v) | 0% | 0.01% | 0.02% | 0.05% | 0.1% | 0.2% | 0.5% |
Figure 2: Fluorescence intensity obtained for cultures expressing the sfGFP protein (bacteria holding the cloned vector) at 0 ; 60 ; 120 and 170 minutes for each condition (0,5% not shown). Are presented in purple, results for a 0.2% glucose ratio; in red, results for a 0.1% glucose ratio; in orange, results for a 0.05% glucose ratio; in yellow, results for a 0.02% glucose ratio; in green, results for a 0.01% glucose ratio and in blue, results for a 0% glucose ratio.
As presented in Figure 2, samples with the highest glucose concentration had a lower fluorescence intensity at all times. Therefore glucose repression of BBa_J04500 is supposed to be functional. Also to mention, bacteria expressing the protein without the GFP fusion showed extremely low or no fluorescence levels (data not shown), confirming the absence of sfGFP in the construction.
From these experiments, we propose that BBa_J04500 can't be used as an IPTG inducible promoter in E. coli D5alpha strain. However, it is still a functional constitutive expression vecter and it can be glucose-repressed to control the protein production.
Conclusion and perspectives
These conclusions mean that the vector can still be used in DH5alpha cells, with specific methods : the promoter must be repressed using glucose, which concentration is linked to protein production as glucose disappearance will trigger it.
An idea for the next contribution of BBa_J04500 could be to characterize more precisely the glucose repression. To do so, it could be interesting to study the link between glucose concentration and the time it takes to see a lifting of repression. It would permit learning how to use the glucose repression capacity of this part efficiently.
Comparison with the Documentation made by ICJFLS 2021
While adding our contribution to this part’s page, we were very interested to see that iGEM ICJFLS 2021 team also chose to work on the characterization of BBa_J04500. Surprisingly, they found out that the promoter responded to IPTG addition, which seems to be in opposition with our own results.
We hypothesize that this could be linked to the use of different E. coli strains in the two teams. We used DH5alpha for our experiments, while the strain used by ICJFLS-2021 team is not specified. Some E. coli strains (like BL21(DE3)) have a LacIq genotype, which leads to the overexpression of the LacI repressor, and might have a tighter regulatory effect on BBa_J04500 than the DH5alpha strain.
In conclusion, the work done by ICJFLS-2021 team and ourself show that more experiments are needed in order to characterize how the promotor behaves in different E. coli genetic backgrounds.