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Revision as of 22:55, 18 October 2016
Results
Promoters
pH Sensing Device
Introduction
Bacteria have the ability to sense their surroundings and respond to them by turning on genetic regulatory systems. One of the environmental stimuli to which bacteria may respond is pH1. Bacteria respond to environmental stimuli for a variety of reasons for example to trigger pathogenesis or survival genes, which help the bacterium, survive, thrive and compete in a new environment1.
We found several promoters that were submitted from previous iGEM teams, this included Pasr (BBa_K1231000) and PgadA (BBa_K1231001) both submitted by the 2013 Northwestern iGEM team. These have been reported to be activated at low pH. So we started by characterising these two promoters and finding the best one suitable for our project. We cloned gfp (BBa_E0840) downstream of both of these promoters in order to measure expression levels (Pasr-gfp (BBa_K1962014) and PgadA-gfp (BBa_K1962013 )). For our bile salt responsive promoter we used PacrRA (BBa_K318514), submitted by the 2012 Wisconsin –Madison iGEM team, which consists of the acrRA operon found in salmonella enterica strain. This operon contains the RamA binding sequence, the acrR gene and part of the acrA gene. We then cloned a gfp biobrick (BBa_E0840) downstream of PacrRA (BBa_K1962010 ). Due to E. coli not containing its own RamA transcription factor we codon optimised the ramA gene sequence for expression in E. coli and got it synthesised by IDT as a gBlock gene fragment. RamA was also submitted by the Wisconsin-Maddison 2012 iGEM team (BBa_K318516) but was out of stock in the iGEM registry so we also submitted this as a biobrick (BBa_K1962009 ).
Pasr
Enterobacteria can respond to low pH by de novo synthesis of specific proteins and altered levels of gene expression. The response to environmental stresses, such as pH, is often a complex mechanism and also depends on other environmental factors such as nutrition, the presence or absence of oxygen or starvation2. The E. coli asr (acid shock RNA) gene encodes small RNA, of about 450 nucleotides in length2. This gene is inducible by low external pH and contributes to the organism’s survival2. It has been suggested that asr genes may be regulated by the two component system PhoBR2.
In this two-component system the protons from the environment (H+) activate the sensory part (PhoR- in the periplasm) of the two-component system, which then transduces the signal to the activator protein- PhoB, which can bind promoter DNA of asr2. The promoter region of asr was analysed and showed to contain a sequence similar to the pho-box; this is a consensus sequence able to bind to the PhoB protein2. These interactions of H+ from the external environment with this two-component system are thought to lead to asr transcription2. Interestingly the PhoBR system also controls the pho regulon which is induced by phosphate starvation, the link between these two factors is not fully understood however, it has been suggested that the level of Asr expressed in minimal media is higher than in enriched media2.
PgadA
Bacteria have a variety of environmental response mechanisms; the GAD (glutamate decarboxylase) system in E. coli has been suggested to be the most effective response to environmental acidic conditions3. This system uses two main isoforms – gadA and gadB and a putative glutamate/Q-amino butyric acid antiporter encoded by gadC3. By decarboxylation of glutamate the protons that leak into the cell can be consumed4. The end product, γ-aminobutyric acid (GABA), is then transported out of the cell by GadC4. The control of this system is very complex involving two repressors (H-NS and cyclic AMP receptor protein), one activator (GadX), one repressor activator (GadW) and two sigma factors (σS and σ70) 3.
Design
The device was designed to be active upon ingestion at a pH of approx. 5.5 or below in order to release our colicins. The promoter is not constitutive and activates transcription of genes at a low pH. The pH range of the promoter was investigated in this project.
In 2013 the Northwestern iGEM team submitted two pH sensitive promoter Pasr (BBa_K1231000) and PgadA (BBa_K1231001). In order to further characterise both of these promoters we cloned gfp (BBa_E0840) downstream of both promoters (Fig 1). We then used this construct to measure and compare the GFP expression levels in response to different pH conditions.
Figure 1: Schematic representation of pH sensitive promoters-gfp constructs in pSB1C3 plasmid.
We wanted to test both our pH sensitive promoters, Pasr and PgadA by monitoring for the production of GFP by western blotting. The western blots showed levels of GFP protein production by the cells at a range of pH values. In Fig 2A the expression of GFP at pH 5 is much higher than the other pH’s. This would indicate that at this pH the largest amount of GFP was being produced under the induction of the gadA promoter. The promoter is slightly leaky as there is also expression of protein within cells at other pH values. In Fig 2B the GFP protein expression under the regulation of Pasr can be seen, the range of activity for this promoter is much wider indicating that this promoter is leakier than PgadA. The level of expression of GFP under the regulation of Pasr appears to be uniform with only a slight increase in GFP at pH5.
Figure 2: GFP production under the control of pH sensitive promoters PgadA and Pasr. Promoters with gfp cloned downstream of them were transformed into MG1655 E. coli cells, 5 ml of these cells were incubated for 16h at 37oC. After 16h, 200 ul of the overnight cultures were introduced into 1.8ml of MOPS pH adjusted LB for the respective pH values indicated above. After 20 min at ambient conditions a 1ml aliquot was pelleted. The pellets were re-suspended in 100 ul Laemmli buffer and 15l samples were then separated by SDS PAGE (12% acrylamide) and transferred to PVDF membrane followed by probing with anti-GFP antibody. Fig 1A - Shows the GFP production under the control of the PgadA and Fig 2B shows the GFP production under the control of the Pasr promoter both in varying pH conditions.
We then compared the results from the western blots to their respective coomassie-stained protein gels. Fig 3 shows the amount of total protein produced by the cells at the respective pH values. The low levels of general protein expression at pH5 (Fig 3A), emphasise the GFP expression observed in Fig 3B at pH5. Likewise, the high general protein expression observed in cells over pH 6 and 7 in Fig 3B lessen the value of the GFP expression observed at this respective pH in Fig 2B.
Figure 3: Coomassie stained protein gels for each pH sensitive promoter-GFP construct. SDS-PAGE gels were loaded with the same samples as shown in Fig 2, and stained with coomassie dye. Fig 3A shows total protein expression in cells containing PgadA-gfp on a pSB1C3 plasmid. Fig 3B shows total protein expression within cells containing the Pasr-gfp construct on a pSB1C3 plasmid. Level of background protein expression gives a clearer overview of the protein expression observed in Fig2A and 2B.
The data gathered from SDS PAGE experiments gave us an overview of the pH sensitive promoter’s optimal pH range, we now wanted to be able to use a more quantitative approach to assign optimal promoter function conditions. To do this we used a 96 well plate reader to monitor GFP fluorescence and cell growth over the space of 20 hours. This experiment took obtained GFP fluorescence and OD600 values every 20 minutes yielding a large volume of data and more quantitative values for promoter function over 20 hours. To normalise flouresence measurements we used the following formula:
GFP / OD600 = Fluorescence per unit absorbance
The data was then plotted to observe the trend over time as well as taking a snap shot at 16h which is the length of time that overnight samples were left before running SDS PAGE experiments as seen in Fig 2 and 3. The data collected from the plate reader experiments showed a slight difference in optimal pH for the pH sensitive promoter asr. As can be seen in Fig 4A and 4B the maximal fluorescence observed from GFP per cell over time was at a pH range of 8 which is significantly higher than what we were expecting from the results observed in figure 3. This may have been due to the cell growth variable of this experiment, as the western blot method did not incorporate this factor and essentially only showed expression after 20 minutes of being exposed to a different pH. For PgadA we observed a coherent trend of optimal promoter function around pH5/pH6 as can be seen in fig 4A and 4B.
Figure 3: 96 well plate reader experiments. Full time frame showing trends in GFP/Cell over 20h. Promoters with gfp cloned downstream of them were transformed into MG1655 E. coli cells, 5ml of these cells were incubated for 16h at 37oC. After 16h, 200ul of the overnight cultures were introduced into 1.8ml of MOPS pH adjusted LB for the respective pH values. 200ul of each pH-cell buffered LB was transferred into each well. A negative control containing no cells and a negative control containing non-buffered fresh LB were also transferred in 200ul samples into the plate. 3 repeats of each variable were carried out.
Figure 4: 96 well plate reader experiments. 16h time snap-shot out of the total 20h which can be seen in figure 3. 16h was the average time of overnight cultures thus this would have been enough time for the cell growth to stabilize and adapt to the conditions.
Bile Salts Sensing Device
Introduction
Bacteria that live in the intestine must be able to withstand the antimicrobial properties of bile salts1. Two mechanisms that bacteria have developed to withstand bile salts are modified membrane structures and efflux pumps to transport bile out of the cell1.
This part of the construct consists of biobrick Bba_K318514 which consists of the acrRA operon containing a RamA binding site, acrR and acrA genes.
Salmonella has multiple efflux pumps, including AcrAB. The transcription of proteins required to make this pump can be induced with the help of a transcription factor called RamA. This transcription factor recognises and is bound to by bile salts and then binds to the RamA binding site on the acrRA operon2. This regulatory binding causes increased transcription of the acrAB gene2. The efflux pump can then begin to remove bile salts out of the cell and thus protect the bacterium from it’s environment1. To apply this concept to the timed lysis mechanism, a GFP reporter (part BBa_E0840) was cloned downstream of it.
acrRA:
The device was then further designed to lyse the cells upon entering the intestine thus to release the colicins produced within the cell during the time spent in the stomach. This was also a design to prevent the release of live GMO into the environment. First we wanted to characterise the bile salts sensitive promoter acrRA (BBa_K318514). This part was submitted by the 2012 Wisconsin – Madison team, which consists of the acrRA operon found in Salmonella enterica. This system requires a transcription factor known as RamA which binds to the acrRA operon and activated downstream genes. ramA (BBa_K318516) was previously submitted by the Wisconsin – Madison iGEM team in 2012, however, we were unable to obtain this part and so codon optimised the ramA gene for E. coli and had this synthesised by IDT as a gBlock gene fragment as submitted it as a biobrick.
We cloned gfp (BBa_E0840) downstream of the acrRA promoter in order to detect changes in gene expression. RamA was cloned into the pUniprom vector downstream of the constitutive tat promoter. RamA was amplified with HA tag in order to detect expression of the protein.
Figure 5: A- PacrRA construct with gfp. B- pUniprom plasmid constitutively expressing RamA-HA.
Results
To test this part, ramA (BBa_K318516), which had been cloned into a pUniprom backbone, in order to ensure constitutive production, was double transformed with the acrRA-gfp in a psb1c3 backbone into E. coli MG1655 cells. These cells now contain one psb1c3 plasmid with acrRA-gfp and one pUniprom plasmid with ramA, the transformation was done on agar plates containing 1x chloramphenicol and 2x Ampicillin. Colonies from the agar plate were streaked on MacConkey agar plates and left over night. They were then imaged with a Fluorescence Microscope to check GFP expression. The LB plates were also viewed under a microscope with a GFP visualisation software to see whether the presence or absence of bile salts affected promoter function.
Figure 6: Microscopy fluorescence imaging of acrRA-gfp with and without RamA transcription factor on LB nutrient agar plates.
Figure 7: Microscopy fluorescence imaging for PacrRA-gfp with and without RamA transcription factor on MacConkey agar plates.
The results from the fluorescent imagine methods in figures 10 through 13 show that when bile salts are present the induction of the promoter is as expected, only when both the promoter and transcription factor, RamA, are present do we obtain high levels of protein expression (GFP). When the transcription factor is absent there is a low level of background GFP expression, which might not necessarily be due to promoter induction. When the same experiment was carried out in LB, figures 6-9, there was a significant amount of GFP fluorescence with and without the promoter being present. The causes of this are still unknown. Following this the western blot in figure 14 also shows the high levels of protein expression of GFP regardless of the transcription factor being present. As mentioned before the causes for this are still unknown.
iii. Western blots from 12% acrylamide protein gel, samples taken from an overnight in LB.
Figure 14: acrRA bile salt promoter anti-GFP western blot. acrRA-gfp in pSB1C3 and ramA in pUniprom both plasmids in MG1655 E. coli. Cells were grown over night in LB. transformed into MG1655 E. coli cells, 5ml of these cells were incubated for 16h at 37oC. After 16h, 200ul of the overnight cultures were introduced into 1.8ml of MOPS pH adjusted LB for the respective pH values. After 20 minutes at ambient conditions a 1ml aliquot was pelleted. The palettes were re-suspended in 100 ul Laemmli buffer and 15ul samples were then separated by SDS PAGE (12% acrylamide) and transferred to PVDF membrane followed by probed with anti-GFP antibody.
ramA
Salmonella has multiple efflux pumps, including acrAB. The transcription of proteins required to make this pump can be induced with the help of a transcription factor called ramA (part BBa_K318516 – Submitted in 2010 by Wisconsin-Maddison). This transcription factor belongs to the AraC family and controls the AcrAB multidrug efflux pump system in Salmonella enterica through dual regulatory modes depending on environmental signals1. Cholic acids bind to ramA and alter the existing structure1 which has then been proposed to activate the acrRA promoter.
Overnight cultures were taken in liquid Maconkey medium and normal LB with 1x chloramphenicol and 1x ampicillin. The optical densities measured, 1ml of these cells was then spun down and re-suspended in the appropriate amount of Lamelli dye ready to carry out a western blot in 12% acrylamide protein gel. This was repeated twice, one western blot was blotted with anti-HA (to detect the HA tag on the RamA) and the other with anti-GFP antibodies to detect GFP expression.
The results collected for ramA can be seen in figures 6 through 14, ramA when cholic acids are present behaves as predicted, (figures 10-13) inducing the acrRA-gfp construct and showing fluorescence. In figures 6-9 the action of ramA is ambiguous as the promoter is induced in the absence of bile salts. The causes for this are unknown. In figure 15 the ramA transcription factor containing its HA tag was not observed, this was due to the amount of ramA being produced by the cells being rather low so in figure 16 the western blot was repeated and successful blotting for ramA observed. The strain used (Rosetta cell line) contains a viral T7 polymerase, activated by IPTG, which increases the transcription of ramA thereby increasing the concentration of it present and allowing the anti-HA antibody to bind sufficiently.
Figure 15: Anti-HA tag western blot for RamA in pUniprom and acrRA-gfp in psb1c3 in MG1655 E. coli cells. Cells were grown over night in LB. transformed into MG1655 E. coli cells, 5ml of these cells were incubated for 16h at 37oC. After 16h, 200ul of the overnight cultures were introduced into 1.8ml of MOPS pH adjusted LB for the respective pH values. After 20 minutes at ambient conditions a 1ml aliquot was pelleted. The palettes were re-suspended in 100ul Laemmli buffer and 15ul samples were then separated by SDS PAGE (12% acrylamide) and transferred to PVDF membrane followed by probind with anti-HA antibody.
Figure 16: pUniprom ramA was transformed into Rosetta cell lines containing a viral T7 polymerase which is activated by IPTG. Anti-HA tag western blot for RamA in pUniprom within the Rosetta cell line (DE3). Cells were grown over night in 5ml LB then sub-cultured into 25ml LB. Once an OD of 0.4 was reached the samples were added to a final concentration of 1mM IPTG, or for samples not containing IPTG, the volume was made up (with LB) to equal the same as all other samples. 1ml of this was pelleted and Lamelli buffer added before loading 15µl samples onto the protein gel. SDS PAGE (12% acrylamide) was run and transferred to PVDF membrane followed by probind with anti-HA antibody.
We then conducted a plate reader experiment to better determine whether the presence or absence of bile salts (in this case Sodium Cholate, which is the salt of cholic acids) would make a significant difference to the activation of the promoter and to test whether the promoter would still be active in minimal media. From figure 17A we can see that the promoter construct (acrRA-gfp + ramA) is active with and without the addition of Sodium cholate however, the activity in its presence is higher. Sodium cholate appears to have an effect but it’s significance is debatable. Figure 17B shows the activity of all constructs in the presence of Sodium cholate and in the absence of the transcription factor (ramA) the promoter is being activated. In conclusion we have discovered that this part is active in nutrient broth, MacConkey agar, Sodium cholate-minimal media broth and minimal media without sodium cholate, we do note that in the presence of bile-like salts the activity of the promoter is increased. The mechanisms of how this works on a molecular level are still unknown.
Figure 7: Figure 17: 96 well plate reader experiment, measuring OD600 and GFP fluorescence over 20h. acrRA-gfp in psb1c3 plasmid with and without ramA in a pUniprom plasmid. Control consists of both empty pUniprom and empty psb1c3. 16h over nights were grown at 37oC and then normalized to an OD of 1 with minimal media. Stock of Sodium cholate (in sterile water) was diluted with minimal media to make up to a concentration of 10µg/m. The control contains no Sodium cholate. 6µl of normalized sample was added to 194µl of either Sodium Cholate minimal media or Sodium cholate free minimal media (control).17A: Figure showing the affect of the presence of Sodium Cholate for the acrRA-gfp + ramA construct.17B: Figure showing the difference in fluorescence per cell when samples are in the presence of Sodium cholate (10µg/ml)
The device was then further designed to lyse the cells upon entering the intestine thus to release the colicins produced within the cell during the time spent in the stomach. This was also a design to prevent the release of live GMO into the environment.
Figure 18: Construct design, acrRA bile salt promoter followed by genes from a lysis cassette taken from Serratia marcescens consisting of chiW and chiX taken from Prof. Seargent’s Lab at Dundee University.
The promoter acrRA was active in LB and this resulted in an inability to clone toxins downstream of it, instead we have greated a simple biobrick. This part is simply ChiW and ChiX (see figure 17) without the acrRA promoter. Alternatively we could have used the pBAD plasmid to properly control expression of these lysis genes however, there was not enough time to try this. The cloning of psb1c3 (chiWX) was in progress during the wiki freeze.
Figure 19: Lysis cassette genes taken from Serratia marcescens consisting of chiW and chiX in a psb1c3 plasmid. (Prof. Seargent’s Lab at Dundee University.)
Design
Introduction
Our idea was to design the constructs with our colicins under the control of our characterised pH sensitive promoters (BBa_K1962013 and BBa_K1962014) and bile salts sensitive promoter (BBa_K1962010). Therefore, in the presence of the chicken GI tract the colicins will be released in the stomach and upon interaction with bile salts in the intestine further colicins would be released. In order to do this we would first need to clone the Colicin Ia/E3/E9 immunity protein with an RBS downstream of the pH and bile salt promoters to protect our producing cell However, we only successfully managed to clone the colicin Ia and E3 immunities (BBa_K1962015, BBa_K1962016, BBa_K1962012, BBa_K1962011). These composite parts were submitted to the igem registry pasr-col Ia-im and pacrRA-col Ia-im.
Detection of HA-tagged immunity proteins: Single E.coli colonies were incubated in 5ml LB overnight at 37°C. 1ml aliquots were pelleted, resuspended in 100μl MOPS 50mM. Then 20μl was added to 20μl laemmli Sample buffer plus B-mercaptoethanol and boiled for 5 minutes. 15μl samples were separated by SDS-PAGE (10% acrylamide) transferred to membrane and probed with HA-antibody. Expression of E3 immunity in front of acrRA with and without RamA transcription factor.
We then attempted to clone in the full length colicin iA in front of its immunity in the Asr and AcrRA immunity, but we were unsuccessful. This could be because the immunity was not being expressed, since we never managed to show its expression via a western blot.
In the future, we would like to clone the colicin iA immunity in front of the GadA pH promoter, followed by the full length colicin iA.
Warheads
The warheads we have selected to add to the MCS of our truncated colicins are toxins Ssp1 and Ssp2 secreted from Serratia.
Ssp1
Ssp1 is an antibacterial toxin secreted from the Type VI secretion system(T6SS) in Serratia marcescens. Bacteria contain numerous independent systems to facilitate protein transport across the cell membrane. Gram negative bacteria have evolved six general secretion systems, type I-VI. Type VI secretion systems span both bacterial membranes, and deliver secreted proteins directly from the cytoplasm into the target cell. They are widespread in nature, present in at least a quarter of all gram-negative bacterial genomes, type VI secretion is a virulence factor for pathogenic bacteria and is important in interbacteria interactions. T6SS in Serratia secretes Ssp1 which is an antibacterial toxin which kills closely related cells, in order to reduce competition. It has peptidoglycan endopeptidase activity, degrading peptidoglycan in the membrane of the target cell, it cleaves between γ-D-glutamic acid and L-meso-diaminopimelic acid in peptidoglycan. The C-terminal toxic domain of Ssp1 has been cleaved and ligated with truncated colicin E9 containing which contains multiple cloning site.
Ssp2
Ssp2 is an antibacterial toxin secreted from the Type VI secretion system(T6SS) in Serratia marcescens. Bacteria contain numerous independent systems to facilitate protein transport across the cell membrane. Gram negative bacteria have evolved six general secretion systems, type I-VI. Type VI secretion systems span both bacterial membranes, and deliver secreted proteins directly from the cytoplasm into the target cell. They are widespread in nature, present in at least a quarter of all gram-negative bacterial genomes, type VI secretion is a virulence factor for pathogenic bacteria and is important in interbacteria interactions. T6SS in Serratia secretes Ssp2 which is an antibacterial toxin which kills closely related cells, in order to reduce competition. It has peptidoglycan endopeptidase activity, degrading peptidoglycan in the membrane of the target cell, it cleaves between γ-D-glutamic acid and L-meso-diaminopimelic acid in peptidoglycan. The C-terminal toxic domain of Ssp2 has been cleaved and ligated with truncated colicin iA and E9 containing which contains multiple cloning site.
Rap proteins are encoded on the same locus as Ssp2, these act as immunity proteins, they neutralise Ssp2 to protect the bacteria from its own toxin.
These are the constructs which we have produced:
The C-terminal of toxic proteins Ssp1 and Ssp2 from s.marcescans were cloned onto multiple cloning sites on the C-terminal of the receptor binding domain of truncated colicins iA and E9, resulting in modified colicins iA Ssp2, E9 Ssp1 and E9 Ssp2. These were fused with a HA-tag then cloned into pBAD18 vector with PBAD promoter of the araBAD operon. PBAD can be repressed with glucose and induced with arabinose.
The modified colicins were ligated with pBAD18 and grown on LB plates with kanamycin resistance and 0.5% glucose, to repress expression of the toxin while it is taken up by cells.
Detection of Ha-tagged Colicin iA Ssp2: Single colonies of ColiA MCS and ColiA Ssp2 2:1 and 3:1 were grown overnight in 5ml LB with 0.5% glucose at 37°C. 250μl of cells were then grown in 25ml of LB at 37°C until they reached an OD of 0.5A. They were then represssed with 0.2% glucose and induced with 0.2% arabinose and 0.5% arabinose and incubated at 37°C for 5 hours. 1ml sample of each was taken, pelleted, then 100μl of laemmli sample buffer and B-mercaptoethanol was added to each and they were boiled for 10 mins. 5μl of pre-induced and induced samples were separated by SDS-PAGE (10% acrylamide) transferred to membrane and probed with HA-antibody. Expression of ColiA Ssp2 was detected when induced by both concentrations of arabinose.
Detection of Ha-tagged Colicin E9 Ssp1 and Ssp2: Single colonies of ColE9 Ssp1 and ColE9 Ssp2 were grown overnight in 5ml LB with 0.5% glucose at 37°C. 250μl of cells were then grown in 25ml of LB at 37°C until they reached an OD of 0.5A. They were then represssed with 0.2% glucose and induced with 0.2% arabinose and 0.5% arabinose and incubated at 37°C for 5 hours. 2ml sample of each was taken, pelleted, then 100μl of laemmli sample buffer and B-mercaptoethanol was added to each and they were boiled for 10 mins. 20μl of samples were separated by SDS-PAGE (10% acrylamide) transferred to membrane and probed with HA-antibody. ColE9 Ssp1 and Ssp2 were expressed when induced with arabinose at both concentrations.
Testing toxicity of modified colicins - Plate Toxicity
Testing toxicity of ColiA Ssp2: A group of colonies of coliA MCS and coliA Ssp2 were added to 1ml LB and OD adjusted to 1.0A, then serial diluted 1/10 6 times. These were then spotted onto 0.2% glucose, 0.01% arabinose and 0.1% arabinose plates. No toxicity was observed.
We decided to repeat the plate toxicity but with a higher concentration of arabinose.
Testing toxicity of ColiA Ssp2 and E9 Ssp1 and Ssp2: A group of colonies of coliA MCS and coliA Ssp2, colE9 Ssp1 and Ssp2 were added to 1ml LB and OD adjusted to 1.0A, then serial diluted 1/10 6 times. These were then spotted onto 0.2% glucose, 0.2% arabinose and 0.4% arabinose plates. No toxicity was observed.
Lysis Experiment
When the plate toxicity tests presented a negative result, we concluded that the modified colicins may not be being secreted by the cell, therefore we lysed the cells and spotted them on MG1655 E.coli to test their toxicity.When the plate toxicity tests presented a negative result, we concluded that the modified colicins may not be being secreted by the cell, therefore we lysed the cells and spotted them on MG1655 E.coli to test their toxicity.
Testing toxicity of ColiA Ssp2 after lysing cells: Cells containing coliA Ssp2 were lysed for 30 seconds using a sonicator. 5μl of the lysate was then spotted onto agar plated with MG1655 cells. ColiA MCS with glucose and arabinose, coliA Ssp2 glucose and PBS were spotted as negative controls, and ampicillin was plated as a positive control. The plates were then incubated at 37°C overnight.
The experiment was repeated, but the cells were sonicated for longer.
Testing toxicity of ColiA Ssp2 after lysing cells: Cells containing coliA Ssp2 were lysed for 1.5 minutes using a sonicator. 20μl of the lysate was then spotted onto agar plated with MG1655 cells. ColiA MCS with glucose and arabinose, coliA Ssp2 glucose and PBS were spotted as negative controls, and ampicillin was plated as a positive control. The plates were then incubated at 37°C overnight.
Testing toxicity of ColE9 Ssp1 and Ssp2 after lysing cells: Cells containing ColE9 Ssp1 and cells containing ColE9 Ssp2 were lysed for 1.5 minutes using a sonicator. 20μl of the lysate was then spotted onto agar plated with MG1655 cells. Empty pBAD vector and PBS were spotted as negative controls, and ampicillin was plated as a positive control. The plates were then incubated at 37°C overnight.
Growth curve comparing the growth of ColiA Ssp2 cells when the toxin is repressed and induced: Overnights of ColiA Ssp2 and ColiA MCS (control) were subcultured in 25ml LB until they reached an OD of 0.67A. 0.2% glucose was added to one culture and 0.5% arabinose was added to the other. These were then incubated for a total of 5.5 hrs, with the OD measured at regular intervals. No significant difference in the growth of those induced compared to those repressed was observed.
Growth curve comparing the growth of ColE9 Ssp1 and Ssp2 cells when the toxin is repressed and induced: Overnights of ColE9 Ssp1 and Ssp2 were subcultured in 25ml LB until they reached an OD of 0.7A. 0.2% glucose was added to one culture, 0.2% arabinose and 0.5% arabinose was added to the others. These were then incubated for a total of 5 hrs, with the OD measured at regular intervals. No significant difference in the growth of those induced compared to those repressed was observed.
Despite showing that our modified toxins are being expressed, we were not able to show that they were toxic to E.coli. We are currently trying to clone another warhead, POW, which is thought to have homology with colicin iA, therefore we believe that it is more likely to have toxic abilities.
References
1: Terry A. Krulwich, George Sachs and Etana Padan,‘Molecular aspects of bacterial pH sensing and homeostasis’, Nature Reviews Microbiology 2011; (9); 330-343.
2. Edita Sužiedėlienė, Kęstutis Sužiedėlis, Vaida Garbenčiūtė, and Staffan Normark1,‘The Acid-Inducible asr Gene in Escherichia coli: Transcriptional Control by the phoBR Operon’, J Bacteriol. 1999; 181(7): 2084–2093.
3. Scott R. Waterman, P.L.C. Small, ‘Identi¢cation of the promoter regions and cs -dependent regulation of the gadA and gadBC genes associated with glutamate-dependent acid resistance in Shigella flexneri’, FEMS Microbiology Letters, 2003; 225: 155-160
4. Marie-Pierre Castanie-Cornet, Thomas A. Penfound, Dean Smith, John F. Elliott and John W. Foster, ‘Control of Acid Resistance in Escherichia coli’, J Bacteriol. 1999 Jun; 181(11): 3525–3535.
