Team:Hong Kong UCCKE/Contribution
Contribution
Additional information on old part: BBa_M36706
We have learnt the following from this 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 (DE3).
In theis study, 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- and rescue strains
Aim:
In our project, iclR deletion (iclR-) is used to maintain the E.coli at a sub-optimal growth rate, so that when positive detection activates the promoter, iclR is expressed to restore the cell’s growth rate. A plasmid enclosing a constitutively promoted iclR is transformed into 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 iclR- E. coli is not a competent cell, a selected colony on the Kanamycin agar plate is suspended in 250μl of calcium chloride transformation solution in preparation of the transformation. 400-600ng of the above plasmid is added to the suspended colony. The following steps are the same as a general transformation procedure.
Results:
The target 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- and iclR-rescue and WT
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-, iclR_KO) and the transformed iclR- with iclR (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 with LB broth, so that there was 200μl of experimental culture per well in a 96-well microplate.
For more information about the cell growth assay, please visit [experiment].
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- 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 iclR in the plasmids when the plasmids are transformed into the E. coli.
Transformation of the MRP gene into two strains of E. coli (Old part: BBa_K3425100)
The recombinant plasmid containing BBa_K3718005 is purchased from IDT. The composite part is shown below:
The plasmid is transformed into DP5a and BL21. DH5a is an E. coli strain which maximises transformation efficiency, while BL21 is an expression strain. .
Since we had encountered difficulties in verifying transformation via cPCR in BL21, team CityU_HK helped us with the following steps. For collaboration between our teams, please go to [Collaboration].
Confirmation of transformation via cPCR:
The results show that the plasmid has been successfully transformed into BL21 and DH5a as the amplified region is 2.6kb. Both colonies’ band size is between 2kb and 3kb, showing that the plasmid is successfully transformed as a result. The extracted plasmid also have the right sizes, aiming at 4.8kb, the band size were between 4kb and 5kb
Expression of MRP:
To report the expression, BBa_E1010 is added downstream of MRP. Expression of mRFP allows a visible signal to be observed when the genes downstream of the lac operon are expressed by the E. coli through the addition of IPTG.
The images from two fields clearly show that mRFP is expressed when IPTG is added, which convinces us that there is a large chance that MRP is also expressed when the operon is activated. This shows the MRP can be probably be expressed under a inducible promoter, in future context of the CadBA promoter. However, SDS-PAGE and western blotting are required to confirm the expression.
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].