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Applications of BBa_J23106
Experience of the Nottingham 2018 iGEM team
Usage and Biology
BBa_J23106 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_K2715106 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_K2715025.
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_J23106 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_J23106 when used in conjugation with a strong RBS shown to function in both Gram-positive and Gram-negative organisms, and the strength of BBa_J23106 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 it’s therefore unlikely to be useful for other future iGEM teams looking to express genes in non-model Gram-positive organisms.
Sheffield 2016 Review
Sheffield 2016 has improved the characterisation of both BBa_J23100 and BBa_J23106. These parts are a strong and medium promoter respectively, that we have used to design our iron detecting device. We have experimentally validated through fluorimetry that there is indeed a significant difference between expression levels of GFP coupled to the strong and medium promoters. Comparative analysis of promoter strengths can be directly interpreted from the data we obtained. This data can be found both on the original part experience pages of BBa_J23100 and BBa_J23106, as well as at our website. Our team has also improved the function of the BBa_J23100 part, by adding an RBS site downstream of the promoter (BBa_K2016005). BBa_J23100 was originally submitted to the Registry by Berkeley 2006.
Fluorescence of JC28 mutants or W3110 wild types transformed with RyhB-GFP constructs under the control of medium (MedGFP) or strong promoters (StrGFP).
Use of this promoter by team Glasgow 2014
were used to express motA and motB together in our composite biobricks:
BBa_ K1463773, BBa_ K1463772, BBa_ K1463770, and BBa_ K1463771 respectively.
These composite biobricks were used to complement the swimming defect of a motA E. coli mutant.
We found that swimming was restored in the following order:
BBa_J23116 > BBa_J23106 > BBa_J23103 = BBa_J23112.
Examination of the sequences of BBa_J23103 and BBa_J23112 showed that they are identical, despite showing different levels of RFP expression in their initial characterisation!
Evaluation of Anderson promoter J23106 in B. subtilis by iGEM-Team LMU-Munich 2012
This Anderson promoter was evaluated without fused RFP with the lux operon as a reporter in B. subtilis. See the new BioBrick BBa_K823008 without RFP and have a look at the Data from the evaluation in B. subtilis.
BBa_K190026 (Planning) constitutive promoter with GVP cluster
BBa_K190028 (Planning) constitutive promoter with GlpF
iGEM-Team Goettingen 2012
Characterization experiment by qrtPCR on BBa_J23100, BBa_J23104, BBa_J23105, BBa_J23106, BBa_J23109, BBa_J23112, BBa_J23113, BBa_J23114 by iGEM Team Göttingen (by C. Krüger and J. Kampf)
We used quantitative real-time PCR as a powerful tool for quantification of gene expression. We used this method to examine the expression rate of the Tar receptor gene under control of promoters from the Anderson family of the parts registry. The BioBricks (K777001-K777008) we used for this experiment can be found here.
The reported activities of these promoters are given as the relative fluorescence of these plasmids in strain TG1 . Promoter constructs were cloned into the vector pSB1C3 and expressed in E.coli BL21DE3 grown in LB-media (lysogeny broth). The measurements were performed for each construct and reference as a triplet. Additionally, we included H2O as negative control to predict possible contamination. For the evaluation of our results, the 2–ΔΔCT (Livak) method was applied. We used the weakest promoter with the lowest expression rate as calibrator for the calculations and as reference the housekeeping gene rrsD of E.coli.
You can find detailed information of the qrtPCR approach here.
Results & Discussion
As mentioned before, both datasets were collected by methods which produce data at different points after the gene expression. Quantitative real time PCR measures the amount of expressed mRNA while relative fluorescence measurements quantify on protein level. In perspective of stability and half-life periods of mRNA and proteins or due to protein modification, it is comprehensible to obtain varying data-sets and expression rates. Another problem that occurred during our quantitative real-time measurements was the deviation in some of biological replicates. This problem was also observed in another group’s experiments (Kelly et al., 2009). They mentioned variations across experimental conditions in the absolute activity of the BioBricks. To reduce variation in promoter activity, they measured the activity of promoters relative to BBa_J23101. Furthermore, the iGEM team of Groningen which participated in 2009 also measured the relative fluorescence of TG1 strain with the promoters J23100, J23109 and J23106 via Relative Promoter Units (RPUs). Their values indicated the comparable tendency to our documented values
iGEM Groningen 2009
We used a number of the constitutive promoter family members for testing our biobricks. The constitutive promoters show the expected level of fluorescence when transformed into E. coli TOP10 cells. Placing parts behind the promoters turned out to be relatively straight forward. We used this part in combination with several biobricks for building our constructs e.g. BBa_I750016 and BBa_K190028 were placed behind the promoters.
UNIPV-Pavia iGEM 2010
The BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J23118 were charcterized in LB and M9 supplemented with glycerol (0.4%) growth media in high copy and low copy vectors in E. coli TOP10 (BBa_V1009).
RPU and doubling time were characterized for all of them, according to the protocols reported below.
The following measurement systems were used for high copy plasmids:
In order to build low copy plasmid measurement systems, the EcoRI-PstI fragment (J231xx-RFP) of each BBa_J61002-BBa_J231xx was assembled into pSB4C5 vector. This fragment contains the constitutive promoter of interest upstream a RBS-RFP-TT expression system.
The following measurement parts were used for low copy plasmids:
The error bars represent the standard deviation for three dfferent wells in the same experiment. Doubling times were evaluated for the described cultures (HC stands for High Copy and LC stands for Low Copy):
It was not possible to evaluate promoters activities in low copy number plasmids in LB because the RFP activity was too weak and not distinguishable from the background.
Discussion: we observed that the ranking previously documented in the Registry is not valid in all the tested conditions, even if a general agreement can be observed. As an example, BBa_J23110 in high copy plasmid is stronger than BBa_J23118, in contrast with the ranking reported in the Registry.
iGEM Team Uppsala University 2012
A promoter test was carried out to put synthetic and natural promoters on the same scale. Every promoter was assembled before B0032-SYFP2 (BBa_K864101) in the backbone BBa_K592200 (very similar to the pSB3x5 backbones). The test was performed in E coli expression strain MG1655 and cloning strain DH5alpha, by flow cytometry fluorescence measurements of single cells.
Triplicates of each strain and promoter were inoculated in 2 mL LB media with spectinomycin (50 µg/mL) and grown overnight shaking at 37° C. Samples were equilibrated in PBS solution at 1:160 dilution for one hour, and then measured by a BD Biosciences FACSaria III. 10^5 cells of each sample were individually measured and averaged, with dead and other non-flourescent cells excluded. Promoter strength is noted as fractions of the reference promoter's, J23101, strength in corresponding strain.
The variance in expression between MG1655 and DH5α may depend on the reference J23101. The maximum protein expression may be lower in DH5α, due to its lower fitness resulting in lower expression of SYFP2 in the J23101 construct. Alternatively, the clone with J23101 in DH5α may have been weaker than average, resulting in higher RPU values compared to other DH5α.
iGEM WHU-China 2013 construction of tandem double promoters
figure 1. The seven different combinations of double tandem promoters,from left to right, are J23106,J23102-J23102,J23102-J23106,J23106-J23102,J23106-J23106,J23106-J23116,J23116-J23102 and J23116-J23106.
We use consecutive promoter BBa_J23106 as a basic part to construct tandem double promoters BBa_k1081003(J23102-J23106),BBa_k1081004(J23106-J23102),BBa_k1081005(J23106-J23106),BBa_k1081006(J23106-J23116) and BBa_k1081008(J23116-J23106).
Data analysis for RPU evaluation
The RPUs are standard units proposed by Kelly J. et al., 2009, in which the relative transcriptional strength of a promoter can be measured using a reference standard.
RPUs have been computed as:
- phi is the promoter of interest and J23101 is the reference standard promoter (taken from the Anderson Promoter Collection);
- F is the blanked fluorescence of the culture, computed by subtracting for each time sample the fluorescence value of a negative control (a non-fluorescent culture). In our experiments, the TOP10 cells bearing BBa_B0032 or BBa_B0033 were usually used because they are RBSs and do not have expression systems for reporter genes;
- ASB is the blanked absorbance (O.D.600) of the culture, computed as described in "Preliminary remarks" section.
RPU measurement has the following advantages (under suitable conditions)
- it is proportional to PoPS (Polymerase Per Second), a very important parameter that expresses the transcription rate of a promoter;
- it uses a reference standard and so measurements can be compared between different laboratories.
The hypotheses on which RPU theory is based can be found in Kelly J. et al., 2008, as well as all the mathematical steps. From our point of view, the main hypotheses that have to be satisfied are the following:
- the reporter protein must have a half life higher than the experiment duration (we use GFPmut3 - BBa_E0040 -, which has an estimated half life of at least 24 hours, or an engineered RFP - BBa_E1010, for which the half life has not been measured, but is qualitatively comparable with the GFP's);
- strain, plasmid copy number, antibiotic, growth medium, growth conditions, protein generator assembled downstream of the promoter must be the same in the promoter of interest and in J23101 reference standard.
- steady state must be valid, so (dF/dt)/ASB (proportional to the GFP synthesis rate per cell) must be constant.
In order to compute the RPUs, the Scell signals ((dGFP/dt)/ASB)) of the promoter of interest and of the reference J23101 were averaged in the time interval corresponding to the exponential growth phase. The boundaries of exponential phase were identified with a visual inspection of the linear phase of the logarithmic growth curve. |}
iGEM HKU 2011
To start characterizing the promoters, we have performed the red florescence intensity measurements for our selected plasmid in the E.Coli MG1655 strain. The data collected is shown below. It is found that promoter J23106 can lead to a higher expression since the fluorescence intensity per OD600 is the highest, while J23103, J23109, J23116 have relative low expression and fluorescence. As our selected promoters have different strength, thus our team is able to use them to fine tune the protein expression.
We used the part BBa_J23106 in the InterLab Measurement context. We assembled a measurement device by inserting the part BBa_I13504 amplified by PCR into the part BBa_J23106. The device was characterized in E. coli NEB10β, JM109, and NEB Express by measuring GFP expression with a cuvette-based spectrofluorometer, a fluorescence plate reader, and a flow cytometer. We also confirmed the promoter activity with a cell-free S30 extract system and measured mRNA by RT-qPCR.
Determination of Noise Levels in Constitutive Promoter Family Members
(characterized by SDU-Denmark 2017)
Fluorescence microscopy and flow cytometry revealed decrease in fluorescence over time for members of the constitutive promoter family.
The expression levels and the noise of four different members of the Anderson promoter collection and their RFP reporter systems, were studied by fluorescence microscopy. These were, in increasing promoter strength, BBa_J23114, BBa_J23110, BBa_J23106, and BBa_J23102
Additionally, the change in RFP expression levels and noise during growth were tested for the promoters with the highest and lowest relative promoter strength by flow cytometry and qualitative analysis by fluorescence microscopy. Combining these two techniques, the expression and noise levels for the promoters were determined as follows:
- The weak promoter, BBa_J23114, exhibited a relatively low expression of RFP, indicating low gene expression and an increasing high level of noise throughout growth.
- Both medium strength promoters, BBa_J23110 and BBa_J23106, displayed a moderate level of both noise and protein expression of the RFP reporter.
- The strong promoter, BBa_J23102, exhibited a comparatively high expression of the reporter RFP and an increasing high level of noise throughout growth.