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Experience of the Nottingham 2018 iGEM team
Usage and Biology
BBa_J23119 is a constitutive promoter constructed by a previous iGEM team. We wanted to establish whether this promoter would be suitable for use in our non-model organism chassis Clostridium difficile, and also wanted to put the strength of this promoter into context by characterising fluorescence using the iGEM Interlab calibration curves, and also compare its strength to the Interlab positive and negative controls. The construct BBa_K2715119 allows us to characterise this promoter more extensively in E. coli with a standardised and reproducible measurement of fluorescence. Additionally this promoter was characterised in C. difficile using the gusA biobrick BBa_K330002 as a reporter gene in place of GFP, as GFP requires oxygen in order to function, and C. difficile is an anaerobic organism. The gusA containing composite used to assay the promoter activity in C. difficile is BBa_K2715027.
In these composite parts we've added a strong RBS BBa_K2715009, shown to function in Gram-positive and Gram-negative organisms, downstream of the BBa_J23119 promoter, driving expression of either GFP or GusA. The composites are part of a family of composite parts which were all characterised in the same plasmid backbone and in parallel in two fluorescence assays, the results of which can be seen below. The positive and negative controls are parts BBa_I20270 and BBa_R0040 respectively, used in the Interlab 2018 study. The composite parts tested in this assay under the same conditions using a range of alternative promoters are as follows:
GusA assay in C. difficile
The gusA containing composites tested in this assay under the same conditions using a range of alternative promoters are as follows:
These two composite parts have enabled a more standardised characterisation of BBa_J23119 when used in conjugation with a strong RBS shown to function in both Gram-positive and Gram-negative organisms, and the strength of BBa_J23119 can now be quantified using the iGEM 2018 interlab units of fluorescence, relative to the Interlab control plasmids, which will hopefully give future iGEM teams a more comprehensive understanding of its strength. We have also shown that this promoter has extremely low levels of activity in the Gram-positive organism C. difficile, and its therefore unlikely to be useful for other future iGEM teams looking to express genes in non-model Gram-positive organisms such as clostridia.
Applications of BBa_J23119
Experience of the IIT_Madras iGEM 2010 team.As submitted on 29th Oct 2010, 0350 IST (+0530 GMT)
The iGEM 2010 IIT Madras team worked with J23119 as a reference in order to characterise a new pH inducible promoter that we have added to the registry this year. While analysing the results of the charactersation experiment we conducted we found that this promoter, in conjunction with an RBS (J23119 + B0034) has upregulated activity at low pH (4.5 - 5).
Experimental DesignThe experiment we conducted involved the use of the construct J23119+B0034 in the plasmid pGL3Basic, which is a promoter-less plasmid that contains Firefly Luciferase as a reporter. The plasmid was transformed into DH5Alpha and cultured as a primary inoculum in Luria Bertani media. Once the inoculum reached an OD600 of 1.0 the culture was used to inoculate pH adjusted media of pH 4.5, 5, 5.5, 6 and 7, all in duplicates. We regularly measured OD600 for these 10 cultures until their OD600 reached 0.5, or reached saturation. We then took 3 independent samples of these cultures with vigorous shaking in-between consecutive samplings.
Observations and InferencesIn this way we obtained a measure of the Luminescence of 30 samples in an arbitrary unit, RLU/s (Relative Luminescence units per sec). The data is as tabulated below.
Table 1 and 2: Data representing experiment done at various pH in duplicate and sampling done in triplicate. The Luminescence has been normalised using harvest-time OD600 readings of each culture.
The data was also plotted, as shown below.
Figure 1: Experimental data plotted for the duplicates showing high level of reproducibility in the data.
From these results we infer that it is highly possible for the constitutive promoter J23119 to be under the control of another stress factor based on pH. In this case it is possible that due to the high pH stress in the medium, the luciferase is over-expressed. It should also be noted that our pH inducible (in the range of 5 - 6.5) did not show this increased activity at low pH.
For more information on our contribution to the world of Synthetic Biology, check out our wiki.
- pGL3Basic vector backbone, as distributed by Promega. http://www.ncbi.nlm.nih.gov/nuccore/13195703, last accessed on 30th Oct 2010, 0350 IST (GMT +0530).
- DH5Alpha E.coli strain. http://ecoliwiki.net/colipedia/index.php/DH5_alpha, last accessed on 30th Oct 2010, 0350 IST (GMT +530).
Experience of CLSB-UK-2016 iGEM team with Part:BBa_K2078002
This consensus promoter was assembled with the reporter protein, blue-purple chromoprotein AmilCP. The construct was carried on the pSB1C3 plasmid for expression and amplification in E-Coli. The plasmid also contained chloramphenicol acetyltransferase for resistance during plating (CmR).
Observations and Inferences
Our E.coli grew slower than other transformants and we were also unable to recover as much plasmid by miniprep. The use of this strong consensus promoter slows down expression times of the AmilCP Part:BBa_K592025 in E-Coli by around 24hours. This image shows a pellet from 5ml LB broth with transformed E.coli and confirms that the part is expressed, but the amount of pellet was much less than we would normally expect to get from 5ml.
The delay in E-Coli growth is also shown by this 24hour streak plate. The chromoprotein is only just becoming visible suggesting that such a strong consensus sequence slows down both the expression time of the part and growth time of E-Coli.
No review score entered. Sriram Srikant
We used j23119 along with the RBS B0034 to provide a constitutive baseline expression during our characterization of p170 promoter cassette. To our suprise we found that under variable ph conditions the expression of the promoter varied, it showed extreme upregulation at low ph (~4.5).
Characterization by K.U.Leuven 2011 iGEM Team
We fused the constitutive promoter to a GFP reporter, and assayed the promoter’s activity after addition of different amounts of arabinose and IPTG. As such these results can serve as a control for the results obtained for the arabinose-inducible BBa_K584000 and IPTG-inducible BBa_K584002 bricks. We tested the activity in a TOP10F’ (Figure 1 & 3) as well as a MG1655 (Figure 2 & 4) E.coli strain background. For more information on E.coli strain descriptions, we recommend the following website: E.Coli_Genotypes.
Apart from a small growth defect and a somewhat lower fluorescence at the 8 hour time point after the addition of 2% arabinose, the activity of the constitutive promoter in TOP10F’ cells is unaffected by arabinose addition, as seen in Figure 1. For the MG1655 cells (Figure 2), however, we see clear effects of adding arabinose at concentrations of 0.2% or 2%, with promoter activities significantly lower than the control condition. This points to interference of high inducer levels with the activity of the promoter and the importance of metabolism of the inducer, as arabinose can be metabolized by MG1655 cells, but not by TOP10F’ cells.
For both TOP10F’ and MG1655 cells, the activity of the constitutive promoter is unaffected by the addition of IPTG (Figures 3 and 4).
Experience of NAU-CHINA-2016 iGEM team--Kejian Shi
To evaluate each site of the Anderson Promoter`s effect on the whole promoter, the NAU-CHINA Team used TASSEL5 to analyse the correlation degree between each mutation and the strength of the promoter.We combine the sequence and the data of strength , the P-Value is calculated using the GLM whose result is showed as the Manhadon. Our purpose is to add information which can help users to mutate the promoter to the suitable one for their teams.
Figure.1:It shows the correlation degree between the each position of the site of the promoter and the strength of the promoter using a series of Anderson promoters to analyse. The abscissa of the figure represents the position of the site of the sequence of the promoter. The position start at "0", so the last of the number of the site is "34". The ordinate of the figure represents the P-Value of the each site. The higher the P-Value is the higher effect of the site is,and the lower strength of the promoter is at the same time. For example ,we will find out that the 24th site reduce the strength of the promoter greatly from the figure.
Figure.2:The left list shows the position of each site of the promoter,the right list contains the P-Value of the each site. Some of the sites do not have the mutation so that TASSEL can not calculate their P-Value.
The two attachments is the sequence data and the strength data used in analysing with TASSEL.