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Team:Exeter/Detection
From 2014.igem.org
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Testing the sensitivity of our promoters to TNT/NG
Abstract
We have designed two promoters based on the NemR-detecting region of the E. coli genome for the purpose of detecting TNT and NG. In our final construct they could be used for the production of our enzymes, or inverted to activate a kill switch. We observed the change in production of the fluorescent protein iLOV at varying concentrations of TNT and NG. We did this by measuring the growth and fluorescence of 200ul cultures on a 96-well plate. We found that 004 appears to express protein constitutively relative to growth, while 007 had increased expression in response to higher levels of TNT/NG.
Introduction
E. coli has a natural system for detecting damaging compounds within the cell in the NemR system; NemR is a regulatory protein that can bind to a specific region within the genome. When bound NemR inhibits the transcription of genes following this region. However, the presence of reactive species, such as N-ethylmaleimide, hypochlorate or nitroglycerin the disulphide bridge is broken and NemR no longer binds to DNA, activating genes after the region. We have created two regulators utilizing sections of this region. 004 includes the entire NemR region in front of our reporter gene, iLOV, while 007 uses a specific sequence known to be the NemR binding site, and places it between the -35 and -10 sections of a high-level constitutive promoter.
We have tested how well our promoters responded to non-lethal doses of TNT and nitroglycerin by observing the change in fluorescence over time in a fresh culture after a set volume of the chemical had been added. A set volume of overnight culture was added to MYE media in a 96 well plate, as well as a volume of TNT/NG. By using a 96-well plate we were able to massively increase the range of concentrations of compounds we were able to test. The information used to find out lethal and non-lethal levels of TNT came from our studies into The Toxicity of TNT and Nitroglycerin to E. coli.
Throughout this experiment several strains of E. coli were used. 004- and 007-Top10 were being tested for their fluorescence in response to TNT/NG. WT-Top10 cells were used as a negative control, to ensure we weren’t recording any natural fluorescence of Top10. GFP-Top10 was used a positive control to ensure we recorded the fluorescence of the cultures correctly. As MYE media was used to grow the cultures it was also used a negative control, to ensure we weren’t measuring any fluorescence from the media.
Experiment One: Is iLOV fluorescent when expressed in E. coli?
This test was carried out on Plate 1. The only recorded use of iLOV within iGEM is by the Glasgow 2011 Team, and it’s functionality within E. coli was not shown. As we are using it as a reporter protein for our promoters we wanted to make sure that it definitely functioned within the organism.
E. coli was grown in MYE media for 27 hours while its fluorescence and optical density was measured. We carried out this test with WT-Top10, iLOV-Top10, GFP-Top10 and pure MYE media.
Experiment Two: Are our promoters functional with and/or without TNT?
This test was carried out on Plate 2. E. coli was grown for 32 hours with the addition of a set, non-lethal volume of TNT (0-10 ul, with steps of 2.5 ul) at t =0. Over this time the optical density and the fluorescence of the cultures were measured. We did this test to examine how our promoters responded to TNT.
Materials
- TECAM 200 PRO microplate reader.
- Grenier 96 well black plates.
- Top10 E. coli
- Top10 (+BBa_K1398004) E. coli
- Top10 (+BBa_K1398007) E. coli
- Top10 (+Constitutive iLOV) E. coli
- Top10 (+Constitutive GFP) E. coli
- 1000 ug ml-1 Trinitrotoluene
- 1000 ug ml-1 Nitroglycerin
- LB Media
- MYE Media
Method
These experiments were carried out on 96 well plates, using a TECAM 200 PRO microplate reader. The Top10 strains were grown on LB media overnight and scanned while on MYE media. Each strain was grown overnight in 10 ml of LB media in a shaking incubator at 37 oC. To create the culture for each well, 200ul of MYE media was mixed with 3 ul of the required strain, as well as a volume of TNT or NG specific to each well. When MYE of LB media was used as a control 200 ul was used. The wells were scanned in the TECAM machine, a process which took around 5 minutes. The cultures were kept at 37oC while this occurred. The plates were then transferred to a shaking incubator (800 rpm), usually for 55 minutes. In cases where plates where run overnight they were left in the TECAM machine and the shaking function was used. Each plate had a different arrangement of cell cultures. The layout of the plate and location of the cultures used in each experiment is listed below.
Plate 1:
- A1-12 Top10
- B1-12 iLOV
- C1-12 GFP
- D1-12 MYE Media
Plate 2:
- A7-9 004, with 10ul TNT
- A10-12 007, with 10ul TNT
- B7-9 004, with 7.5ul TNT
- B10-12 007, with 7.5ul TNT
- C7-9 004, with 5ul TNT
- C10-12 007, with 5ul TNT
- D7-9 004, with 2.5ul TNT
- D10-12 007, with 5ul TNT
- E10-12 Constitutive iLOV, with 0ul TNT
- F10-12 Top10, with 0ul TNT
- G7-9 004, with 0ul TNT
- G10-12 LB, with 0ul TNT
- H7-9 007, with 0ul TNT
- H10-12 MYE, with 0ul TNT
Results
Experiment One: Is iLOV fluorescent when expressed in E. coli?
Figure 1 shows the fluorescence of WT-cultures, as well as cultures expressing GFP and iLOV. The fluorescence of iLOV (at excitation = 440 nm, emission = 520 nm) reaches a 4400 at 20h, while WT-Top10 cultures at 20h have a fluorescence of 880.
Experiment Two: Are our promoters functional with and/or without TNT?
Figure 2 shows the fluorescence of each cell culture in comparison to its optical density. As the concentration of TNT increases the Flu:OD of 004-Top10 stays fairly consistent, varying between 900 and 1120, while the Flu:OD of 007-Top10 increases as TNT concentration increases, from 380 at 0ul to 1020 at 10ul. The values for fluorescence and optical density were taken at 24 h.
Figure 3 shows the Flu:OD of each culture at each level of TNT in comparison to the Flu:OD at TNT = 0ul. This figure makes it clear that 007 increases in relative fluorescence as TNT concentration increases; eventually reaching 2.7x its original value. The values for fluorescence and optical density were taken at 24 h.
Discussion
Experiment One: Is iLOV fluorescent when expressed in E. coli?
The fluorescence of iLOV in E. coli can clearly be seen in this experiment. There is clearly a huge increase in fluorescence compared to WT-Top10. It can also be seen that under regular conditions maximal iLOV fluorescence is reached between 20-25 hours. This information was used when selecting a time point for the comparison of fluorescence to optical density. The relative fluorescence of GFP and iLOV are not comparable in this experiment as they are under the control of different promoters, although it is suspected that the fluorescence of GFP is greater.
Experiment Two: Are our promoters functional with and/or without TNT?
As shown in “E. coli’s response to Xenobiotic Compounds” cell growth decreases in response to increasing concentrations of TNT. As cell growth decreases protein expression also decreases, proportionally. Therefore it is difficult to associate an increased promoter response with an increased level of TNT. The easiest way to show this data is to present fluorescence relative to the optical density of the culture, which is roughly proportional to growth.
The expression of 004 remains fairly constant relative to growth, while 007 increases in relative activity as the concentration of TNT increases, indicating that it is responding to the increased levels.
004 appears to be a general constitutive promoter, but 007 appears to have a definite response to TNT over other promoters. Although expression appears to slightly increase between 0, 2.5, 5 and 7.5 ul TNT, there is a steep difference between the expression shown in 7.5ul TNT and 10ul. This may indicate a certain cut-off point at which the promoter activates.
Conclusion
iLOV presents a viable option for use in E. coli, as its fluorescence is much greater than that of E. coli. Of our two promoters, 004 appears to have no specific response to TNT, while 007 did appear to have a relative increase in expression as TNT concentration increased. This suggests to us that with some modification it could form the basis of biosensor for TNT, or some part of a regulatory system.
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