Inducible expression of cell-growth-rescue genes and GFP

The genes arcA, iclR, and gfp are expressed under the control of Lac operon. The addition of IPTG will induce the expression of the three genes simultaneously. Different RBS are used to avoid repeated sequence.

Effect of iclR and arcA knockouts on biomass formation and metabolic fluxes in Escherichia coli K12 and its implications on understanding the metabolism of Escherichia coli BL21 (DE3)

In the study ‘Effect of iclR and arcA knockouts on biomass formation and metabolic fluxes in Escherichia coli K12 and its implications on understanding the metabolism of Escherichia coli BL21’, experiments were done on the three strains, which are the single knockouts arcA- and iclR- and the double knockout arcA-iclR-. The three strains are produced from K12 MG1655. The knockout strains were compared against the BL21 DE3 strain in terms of their metabolic flux, especially the flux of carbon molecules through different pathways.

ArcA is a protein that regulates genes that take part in various metabolic pathways as a global regulator, while IclR is a protein that controls the transcription of the glyoxylate pathway under the aceBAK operon as a local regulator.

iclR- and arcA- had a maximum growth rate reduction of up to 13%, while arcA-iclR- exhibits a reduction as much as 38% compared to wild-type parent E. coli in batch culture conditions, where glucose is abundant and limited.

arcA-iclR- exhibits an increased biomass yield of 0.63 c-mole/c-mole glucose under glucose abundant conditions, with the maximum theoretical yield of about 0.65 c-mole/c-mole glucose. There also is an observed decrease in acetate formation and CO2 production. There is an increase in the transcription of glyoxylate enzymes, meaning that the pathway is activated despite the absence of crp-activation under glucose limiting conditions. Moreover, the transcription of TCA genes are liberated in arcA- which causes a higher flux in the TCA cycle, causing lower acetate formation. Flux in the glyoxylate pathway is further increased in iclR- under glucose limiting conditions, but this effect was not observed in arcA-iclR-.

Bacterial growth assay of iclR KO and rescue strains

Aim:

In our project, iclR deletion (iclR-) is used to maintain the E.coli at a non-optimal growth rate, so that when positive detection activates the promoter, iclR is expressed to restore the cell’s growth rate. Transformation of a plasmid enclosing a constitutively promoted iclR is done to ensure recombinant iclR can be expressed in iclR- E. coli (iclR-Rescue). To observe and compare the cell growth dynamics of wild type E. coli, iclR-, and iclR-Rescue, a cell growth assay is done.

Transformation of iclR gene into iclR KO strain

The recombinant plasmid containing the iclR expression unit (BBa_K3718007) is synthesized by IDT.

The above plasmid is transformed into is an iclR- E. coli. The iclR- E. coli is from the Keio collection and is not competent with delivery. Since the strain is non-competent, 400-600ng of plasmid is used to transform it into 250μl of CaCl-suspended iclR- E. coli colony. The other aspects of the transformation is the same as the one detailed in the general protocol. Below is a gel picture from a colony PCR done to verify the transformation.

Results:

The amplified region is approximately 1.1kb. The band sizes of all colonies are between 1 and 1.5 kb, which reassures the desired plasmid has been successfully transformed into the iclR- E. coli.

Cell growth assay of iclR KO and rescue, wild type and arcA KO strains

This is done to compare the growth rates and maximum cell densities between the four strains of E. coli to each other. The three strains, K-12 BW25113 (WT), Keio collection iclR-knockout (iclR_KO) and the transformed iclR_KO with iclR gene (iclR rescue) are grown in starter cultures for 2 hours in which 50μl from the overnight cultures are grown in it to ensure the cells are going through their exponential phase when the experiment starts. Every strain’s starter culture then has its optical density measured, diluted to optical density = 0.05, and is topped up with 200μl LB broth per well in a 96-well microplate.

The data range is quite large for all three strains, causing difficulties in drawing a meaningful conclusion. The results could be improved by more experiments and testing. From the plots below, we will discuss the general trends we saw from the experiment.

The picture above shows how the box plot represents the data. The box represents Q1-Q3, while the whiskers represent Q1-1.5xIQR and Q3-1.5xIQR. The dots represent outliers that are too abnormal and are not included into the whiskers as a result.

Results:

This graph above shows the maximum growth rate between the three strains of E. coli. The maximum growth rate of iclR rescue has a higher growth rate than iclR KO and wild type, and the difference is relatively large for both strains when compared to the iclR rescue strain. The results were not what we had expected, as we expected that the iclR rescue would only have its growth restored to wild type levels, while this shows that the iclR rescue has exceeded the growth rates of the wild type too, which may be due to the cells having multiple copies of the iclR in the plasmids when the plasmids are transformed into the E. coli.

References

H. Waegeman, J. Beauprez, H. Moens, J. Maertens, M. De Mey, M. R. Foulquié-Moreno, J. J. Heijnen, D. Charlier, and W. Soetaert, “Effect of ICLR and ARCA knockouts on biomass formation and metabolic fluxes in escherichia coli K12 and its implications on understanding the metabolism of escherichia coli BL21 (DE3),” BMC Microbiology, vol. 11, no. 1, p. 70, 2011.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 1567
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 2468