Difference between revisions of "Part:BBa K3718007"

Revision as of 16:59, 16 October 2021

This is an expression unit for expressing the protein IclR. BBa_M36706 codes for the protein IclR. It is expressed constitutively under the strong promoter BBa_J23100.

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.

Reference

X. Dong, “Whisker of boxplot: R-bloggers,” R-bloggers, 26-Nov-2013. [Online]. Available: https://www.r-bloggers.com/2012/06/whisker-of-boxplot/. [Accessed: 13-Oct-2021].

Sequence and Features