Improvement by JNFLS2019

Overview:

Plac is one of the most common promoter in life science research field. It is mainly composed of Lac operon containing LacO site. LacI repressor, encoded by LacI gene, can bind with LacO site to inhibit the binding of RNA pol to the promoter, so the genes downstream expression are blocked. Serving as inducer, IPTG can bind with LacI inhibitor, making the latter’s conformation changes, so LacI is detached from LacO site, which enables the transcription of downstream genes. BBa_K741002 is a GFP generator driven by Plac promoter, however there is no LacI gene in it. Although the E.coli could express some LacI, it is not enough for inhibition GFP expression. So this GFP generator has some leakage expression, like the designer stated, even some GFP express without IPTG inducer presence. We constructed a new GFP generator (BBa_K3209006) also driven by Plac promoter. It contains LacI gene, which can lower significantly the leakage expression. Both LacI and EGFP are linked to the downstream of Plac, which is regulated by LacI inhibitor and IPTG inducer. Using EGFP as a reporter, its fluorescence intensity appears a lower leakage expression. This new GFP generator could be self-regulated because LacI protein can inhibit its self expression, so that no excessive LacI expression which is considered as waste of resources. We detected the response of this generator to different concentration of IPTG, indicating that it could be inhibited by LacI, and induced well by IPTG.

Results:

First, we compared the inducing effect on the two GFP generators, using different concentration of IPTG. We set 4 groups: 2 experimental groups including old GFP generator (BBa_K741002) and new GFP generator (BBa_K3209006), one negative control without GFP expression and one positive control with constantly GFP expression. At 0h, all groups’ OD600 approximately reaches to 0.8, then certain concentration of IPTG was added to the culture medium, incubated cells at 22℃ overnight. Measure the fluorometric value at 510 nm and OD600 value for each group every 1h, using an automatic microplate reader.

Figure 1. The inducing effect of IPTG on the two GFP generators. Relative fluorescent intensity is fluorescence per OD600 standardized with fluorescence per OD600 value of each test group at time = 0, IPTG=0. The figure indicated that both BBa_K741002 and BBa_K3209006 expressed GFP induced by IPTG, and different concentration of IPTG had same inducing trend.

Then we detected the GFP expression under the IPTG existance or absence, using BBa_K741002 and BBa_K3209006, respectively. The results showed that BBa_K741002 expressed a mountain of GFP whether the IPTG present or not, which means that BBa_K741002 had a high leakage expression and lower sensitivity to the IPTG induction. However, BBa_K3209006 expressed a high level GFP only under IPTG existance. The leakage expression is very low, and it is sensitive to the IPTG induction.

Figure 2. The comparison of GFP expression using BBa_K741002 with and without IPTG induction. Relative fluorescent intensity is fluorescence per OD600 standardized with fluorescence per OD600 value of each test group at time = 0, IPTG=0. This figure indicated that the GFP generator (BBa_K741002) had a high keakage expression, and low sensitivity to the IPTG induction.

Figure 3. The comparison of GFP expression using BBa_K3209006 with and without IPTG induction. Relative fluorescent intensity is fluorescence per OD600 standardized with fluorescence per OD600 value of each test group at time = 0, IPTG=0. This figure indicated that the new GFP generator (BBa_K3209006) is very sensitive to the IPTG induction, and the GFP leakage expression is very low.

Protocol:

1. Transform the plasmids into BL21 strain.
2. Pick up a single colony by a sterile tip from each of the plates for all the experimental and control groups. And put the colony into 5ml M9 medium with 100 µg/ml ampicillin. Incubate at 37℃ in a shaker for 6-8h.
3. Detect OD600value of the culture medium with spectrophotometer, and dilute the culture medium to OD600= 0.8.
4. Add 100 µl bacteria culture medium into a sterile 96-well plate. IPTG is added to final concentrations of 1, 2, 5, 10mM. M9 medium is the blank control. Constantly expressing EGFP colony is the positive control and colony without EGFP expression is the negative control.
5. Incubate at 22℃ overnight, and measure the fluorometric value at 510 nm and OD600value for each well every 1h, using an automatic microplate reader.
6. The experiment should be repeated at least 3 times.

References:

1. Szabolcs Semsey, Sandeep Krishna.The effect of LacI autoregulation on the performance of the lactose utilization system in Escherichia col, Nucleic Acids Res 2013 Jul; 41(13): 6381–6390.
2. Adam J. Meyer, Thomas H. Segall-Shapiro, Emerson Glassey, Jing Zhang & Christopher A. Voigt. Escherichia coli “Marionette” strains with 12 highly optimized small-molecule sensors. Nature Chemical Biology, 2019, 15: 196–204.

UIUC_Illinois 2019 Characterization

Overview:

We characterized this part using fluorescence at different concentrations of IPTG to determine the level of IPTG we would need to use to induce genes in the system we were testing. The plasmid was transformed into two cell types: NEB 10-beta and a cell strain containing lacIQ. Cell cultures were grown up overnight in 5 mL of LB with chloramphenicol at 37 C. The following day, 990 uL of LB, 10 uL of cell culture, and 1 uL of chloramphenicol were added to 1.5 mL Eppendorf tubes. The tubes were incubated for 4 hours at 37 C. After 4 hours, IPTG of varying concentrations was added to the tubes. Five different conditions were tested, with each condition tested with 4 replicates. The tubes were then incubated at 37 C for 16 hours. 200 uL aliquots were then taken from each tube and spun down for 3 minutes. The cells were resuspended in 200 uL of PBS buffer. 100 uL of resuspended cells were plated on a clear 96 well plate and fluorescence and OD600 were measured. The fluorescence settings were excitation set 485 nm, emission cutoff at 495 nm, and emission at 525 nm.

This graph shows the normalized fluorescence of both tested cell types for all concentrations of IPTG tested. In regular NEB 10-Beta, the expression of GFP is very leaky as expression is high even in samples where no IPTG was added. The lacIQ+ cell line shows overall much lower levels of expression, but is more responsive to the addition of IPTG, with 0.1 mM IPTG providing the greatest increase in expression. These results indicate that pLac-regulated expression is highly leaky in cells that don’t have a lacIQ inhibitor.