Difference between revisions of "Part:BBa M36706"

Revision as of 17:04, 16 October 2021

Gene encoding IclR

IclR represses the aceBAK promoter in E. coli. In the presence of pyruvate, IclR binds the aceBAK promoter regions more tightly and thus the downstream genes are highly inhibited. Therefore when pyruvate is present in high concentrations, the aceBAK promoter should be highly inhibited and downstream gene expression should be low. When pyruvate levels are low, the aceBAK promoter should be less inhibited and the downstream genes should be expressed at higher levels.

Bacterial growth assay of iclR- and rescue strains (Team: Hong_Kong_UCCKE 2021)

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 into iclR-

The recombinant plasmid containing BBa_K3718007 is purchased from IDT. The composite part is shown below:

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.

Sequence and Features

Reference

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.