Contents
Overview
There are many contributing factors that could explain outlier data points for such studies as the 2017 InterLab study and so we decided to investigate two of these. They were the use of commercial derivatives of DH5α and the appearance of plasmid dimers. Commercial strains of E. coli although closely related to DH5α often have slightly differing genotypes. For the purposes of our investigation we used NEB5α and found that expression levels were in the most part higher in NEB5α than in DH5α suggesting that commercial derivatives of DH5α should be avoided for studies such as the InterLab. Plasmid dimers are caused by two plasmids joining together to form one larger plasmid. They are often an issue seen in DH5α as they form spontaneously and are often not screened for. Our results showed the unpredictable nature of dimer behaviour and highlighted the importance of avoiding these in quantitative studies of gene expression as they can cause errors in results.
Introduction
- Aim 1
- Sub-aim 1
- Sub-aim 2
- Aim 2
- Sub-aim 1
- Sub-aim 2
Materials and Methods
Transformations
These were carried out as per our standard calcium chloride protocol.
Calibration
Both the OD600 reference point and the fluorescein fluorescence standard curve were carried out as per the InterLab 2017 Plate Reader Protocol
Dimerisation
To generate dimer plasmids a strain of E. coli JC8679 [2] known to cause interplasmid recombination at a higher frequency and so cause multimers was used. Plasmid DNA of each InterLab device was transformed into JC8679 using standard transformation protocol and was then repurified from JC8679 by normal QIAGEN DNA miniprep protocol. The plasmid extracts were then separated by standard gel electrophoresis and the DNA band corresponding to the dimeric plasmid DNA was cut out from the gel and extracted by standard gel extraction protocol a diagram of the gel showing the multimerisation versus the standard monomer plasmid can be seen in figure 2. Multimerisation didn’t occur 100% of the time so this needed to be repeated until dimers were obtained for all of the test devices and the two controls. This extract was then transformed into DH5α E. coli.
Measurements
For the purpose of the measurements two biological duplicates (colonies) of all 6 test devices transformed in to DH5α, along with a positive and negative control, were grown up in LB broth overnight at 37C with shaking at 220rpm. These were then diluted down to an OD600 of 0.02 and 100 l of the samples were placed into wells of a clear bottomed black sided 96 well plate as shown as per figure 3. This was placed into the plate reader at 37C with shaking at 200rpm and measurements of OD600 and Fluorescence at an excitation of 485nm and emission of 530nm were taken at time points of 0hrs, 2hrs, 4hrs and 6hrs.
Results
NEB5α
To compare the expression levels in the NEB5α strain to the DH5α strain, the test devices were transformed into NEB5α following the same protocol as with the DH5α and measurements of fluorescence and OD600 taken the same as with the DH5α and compared to those results above. As there is a possibility the two strains can have different growth rates it is important to only look at the OD600 corrected results, these are shown in figure 7 below. All of the test devices, except one, showed higher noise when in the NEB5α strain, the negative control being almost 5 times higher in NEB5α, the positive control, test device 1 and test device 2 all being between 5% and 10% higher than their DH5α counterparts, test device 4 being around 35% higher and finally test device 5 and 6 being over 100% higher. As for test device 3, this showed as only having less than 20% of the fluorescence of its DH5α pair.
Discussion
NEB5α
From our results, it seems that the NEB5α and DH5α E. coli have different expression levels with some of the test devices showing double –or in some cases more than double- the expression when in NEB5α and so NEB5α isn’t suitable when attempting to compare results to DH5α. However, it isn’t clear where the differing in expression is likely to have come from and so more analysis of this strain needs to be carried out and we would advise that in future InterLab studies only the DH5α strain should be used and no commercial derivatives should be used instead as these cause alterations to the results.
Dimers
References
- ↑ Kiliç, A. O., Pavlova, S. I., Ma, W. G. & Tao, L. 1996. Analysis of Lactobacillus phages and bacteriocins in American dairy products and characterization of a phage isolated from yogurt. Appl Environ Microbiol, 62, 2111-6.
- ↑ Summers, D.K. & Sherratt, D.J. Resolution of ColE1 dimers requires a DNA sequence implicated in the three-dimensional organization of the cer site. EMBO J 7, 851-8 (1988).
