Team:Lund/Experiments
Introduction
The laboratory work consisted mainly of two different parts. The first part was the cultivation of E. coli for curli production which includes five different experiments and the relevant protocols can also be found in this section. The second part consisted of the transformation and subsequent protein production. Several trials and experiments were carried out in order to achieve a successful result. The process that was followed is stated in detail in this page and together with the corresponding protocols.
Curli
4100 .coli is the main producer of curli in the gut and according to literature, one of the producing strains is E. coli MC4100. This strain is able to produce curli at 37ºC [1] when incubated in vitro, corresponding well to approximately the body temperature.
To investigate the effect of the inhibitors it is important to know the amount of curli E. coli produces. Usually, E. coli curli producers produce cellulose, aiding the formation of the extracellular matrix. Since our focus was exclusively curli and not cellulose, another reason to choose E. coli MC4100 strain is because this strain does not produce cellulose.
MC4100 bacterial cells were grown at 37ºC with liquid LB medium without shaking conditions to avoid disrupting curli fibers. The presence of curli was monitored at different cultivation times, up to 8 days after a protein purification specific for curli, where the cells were sonicated at a slow frequency to separate curli fibrils from the cell surface where they attach.
Since curli production was deficient at 37ºC, different conditions were tested, for instance, its growth temperature was shifted to 26ºC in order to stress the cells and therefore try to enhance curli production. Shaking conditions were also assessed without success.
Curli fibrils formed by CsgA monomer can be detected by amyloid-specific dyes such as Thioflavin T (ThT), where the detection of amyloid fibers is based on fluorescence. An enhancement of the ThT fluorescence is produced by binding to amyloid fibers with maximum excitation and emission at 450 nm and 480 nm respectively.
However, a number of different conditions can affect the fluorescence intensity when using ThT as a detection method and it needs to be taken into account when processing the sample. Some of the factors that can affect are; the specific protein forming the fibril, ThT concentration, pH, fibril morphology and ionic strength, among others [2]. Additionally, in all the reactions where ThT binds to macromolecules, the Brownian motion (which is the random motion of particles suspended in a medium) of the ThT is altered. This can be detected by an increase in fluorescence anisotropy, a phenomenon where the light emitted by a fluorophore has unequal intensities along different axes of polarization [3].
For the experiments conducted to detect curli, the used concentrations of ThT were 10 µM and 40 µM according to literature. More than 40 µM is not recommended because ThT becomes self-fluorescent and it needs to be corrected [4].
Due to all the possible factors that can influence the fluorescence when using ThT for curli detection, the experiment was designed to have three different samples by changing conditions in order to acquire a valid method.
The sample treatment before the addition of ThT is outlined in Figure 1. The first sample tested with ThT was a direct sample from the culture, containing the cells that are producing the curli [1]. Thereafter, a second sample consisting of the supernatant of the culture after sonication with an ultrasonic water bath was taken. The curli fibers which are attached to the surface of the cell were released to the medium upon sonication of the culture; and the cells were removed by centrifugation. This sample should contain the curli fibers but without cells [2]. Finally, the third sample was the protein solution, where the second sample was centrifuged at higher speed so the fibrils were present in the pellet and resuspended in Phosphate Buffered Saline (PBS) [3].
Figure 1. Sample treatment before addition of ThT. The numbers refer to the sample cited in the text.
Additionally, in order to not only measure the presence of curli but also at what point the cells start producing it, samples from several cultures were taken at different times of cultivation and ThT measurements were performed for all the samples.
The first experiment consisted of measuring the fluorescence with 10 µM and 40 µM of ThT of 6 samples at different cultivation times (See Table 1).
Table 1. Cultivation time for 6 different samples for experiment 1.
|
Sample |
1 |
2 |
3 |
4 |
5 |
6 |
|
Cultivation time |
2 h |
9.5 h |
21.5 h |
33.5 h |
45.5 h |
57.5 h |
For all those samples, no ThT fluorescence was detected.
A possible explanation for this is that there was CsgA produced, but as the ThT is used to detect amyloid fibers, there might not have been enough time to produce curli fibers. Also, the second sample (2) (See Figure 1) had an SDS treatment, but this step was avoided for the following experiments since the SDS could denature the curli proteins and give incorrect readings. Together with other changes, the new sampling that was followed as in Figure 2.
Figure 2. Sample treatment before addition of ThT in new protocol.
For these reasons, a second experiment was conducted and 8 samples were taken at increased cultivation times and avoiding the SDS step (See Table 2).
Table 2. Cultivation time for 8 different samples for experiment 2.
|
Sample |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
|
Cultivation time |
41 h |
62.5 h |
86.5 h |
111.5 h |
135.5 h |
159 h |
183 h |
207 h |
In addition, a sample after 207 h of cultivation was taken from a MC4100 culture at 26ºC to see if the temperature was affecting the curli production.
Since the ThT results were not clear, a Bradford assay was performed with the supernatant after centrifugation of the cells for the 207 h experiment samples. The results were not conclusive; it was expected to have an increased amount of proteins as the cultivation time was increased, but the results did not show that.
To make sure that there was not any problem with the concentration of the sample, two of the samples with higher amounts of proteins, from the Bradford results (without taking into consideration the cultivation times) were left open for evaporation overnight.
Additionally, a new testing for the ThT was performed to see if adding it in the culture before taking the samples could affect the results differently.
The results for both samples were not conclusive; it was not possible to detect a specific peak for curli when measuring the fluorescence of ThT in the spectrofluorometry.
In pursuit of a clear result of absence or presence of curli, bromophenol blue (BPB) was used, as according to the literature this dye can be used in amyloids [5]. The results of this experiment were not conclusive as not enough intensity was observed to be indicative of the presence of curli.
As the previous experiments did not work as expected, the protocol followed in the previous experiments was slightly changed. The main difference is that the whole culture is centrifuged and the pellet is resuspended in PBS, before the sonication, to have everything in a smaller volume.
A new experiment was carried out, experiment 3. Apart from the introduction of the new protocol, the sonication method is changed to improve the release of the fibers attached to the cells. An ultrasonic probe is used. To test if the cells remain intact after sonication using the probe, survival tests are performed by spreading cell samples on LB plates and checking for growth.
For experiment 3, samples from two temperatures and two cultivation times were measured with ThT, using an ultrasonic probe (See Table 3).
Table 3. Cultivation times and temperature for different samples for experiment 3.
|
Sample 1 |
Sample 2 |
Sample 3 |
Sample 4 |
|
|
Cultivation time (h) |
95 |
95 |
114 |
114 |
|
Temperature (ºC) |
26 |
37 |
26 |
37 |
For this experiment the sonication method used was 2x1min at 60% with the ultrasonic probe. As it did not show any expected peak when measuring the ThT fluorescence, a following experiment was performed by changing either the time or the intensity to see which method could be used onwards.
Experiment 4, was conducted by changing the volume of PBS where the centrifuged samples are resuspended and two sonication methods with an ultrasonic probe were tried:
- Method A: 2x2 min at 60% intensity
- Method B: 2x1 min at 80% intensity
Both sonication and the PBS volume modifications were applied in two different cultures of E. coli MC4100 grown at 26ºC after 138 hours of cultivation. The sample from the first culture was resuspended in 5 mL of PBS and method A of sonication was used. For the sample of the second culture, 10 mL of PBS was used. This culture was divided into two: 2A and 2B. The sample 2A was sonicated with method A and the sample 2B was sonicated with method B.
For experiment 4, survival tests were done with all the cultures before and after sonication. MeThose results indicated that method A and B were correct and they did not disrupt the cells. But when measuring the ThT fluorescence, no peak was observed.
A final experiment, 5, was performed involving other E. coli strains that are also curli producers to compare them with our main strain, E. coli MC4100. For this purpose, three different cultures were used. A BL21 (DE3) E. coli strain was used for the first culture and was cultivated at 26 ºC for 143 h. A TG1 E. coli strain was used for the following two cultures; the first one was cultivated at 26ºC and the second one at 37ºC, both for 47 h. Samples from those three cultures were taken (Figure 2) to measure ThT fluorescence.
At the same time, fluorescence measurements for two more cultures of MC4100 E. coli cultivated at 26 ºC for 138 h were performed. In this experiment, the sonication method B was used.
DNA and Cloning Work
To read more about the system design and the cycles we divided the work into, see Engineering.
General Workflow
Below are listed protocols that concern the cloning process. These were used in the Building phase of the three engineering cycles, and always occur in sequence for each sample/experiment. If Gibson assembly is used for cloning, the sequence is as follows:
Plasmid preparation → Gel electrophoresis → Purification of plasmid from gel → Digestion of plasmid → PCR amplification of inserts → Purification of PCR product → Gibson assembly → Transformation into cloning host→ Colony PCR → sequencing → Plasmid purification → transformation into expression host
In restriction enzyme cloning, the sequence is nearly identical. However, in the step “Digestion of plasmid” a dephosphorylation step is added and the step “Gibson assembly” is replaced with “Ligation”.
Before the cloning starts, several preparations are needed, including making competent cells, preparing enough LB liquid and solid medium with or without antibiotics.
For detailed information about experimental procedures, see protocols at the end of this page.
Throughout the project, the different experiments were repeated and adjusted as the results were interpreted. For example, a temperature gradient was used in the PCR amplification of inserts to find the optimal annealing temperature. Another example is the extensive tests that were performed for transformation using only intact plasmid and a negative control to check if the protocol was working and to optimise the antibiotic concentration.
Plasmids and Strains in Cloning
Two plasmids were used in our project; In cycle 1 and 2 we used pTRKH3-ermGFP, gifted to us by Michela Lizier (Addgene plasmid # 27169 ; http://n2t.net/addgene:27169 ; RRID:Addgene_27169), and in cycle 3 we used pET-11a, gifted to us by our supervisor Johan Svensson Bonde.
In all three cycles, two strains of E. coli were used as cloning hosts: E. coli TG1 and E. coli XL1. In cycle 1 and 2, when pTRKH3-ermGFP was used, neither strain had consistent differences between positive and negative results, but E. coli XL1-blue was mainly used. In cycle 3, when pET-11a was used, E. coli TG1 was mainly used as formed more colonies on positive control plates for transformation as compared to E. coli XL1-Blue.
Inhibitor Expression
Following the cloning procedure described in the workflow above and the protocols below, the inhibitors can be expressed in an expression host. In cycle 1 and 2, the plan was to use our chosen chassi, L. reuteri, as expression host, which pTRKH3-ermGFP previously has successfully been utilised for [6]. The CP44 promoter used in these cycles is constitutive [7], therefore no inducer would be used. Due to unsuccessful transformations using pTRKH3-ermGFP, L. reuteri was never transformed or cultivated in our project.
For pET-11a, the plasmid used in cycle 3, the expression host used was E. coli BL21(DE3). pET-11a utilizes the T7 expression system, which requires a host which can produce T7 RNA polymerase. T7 RNA polymerase originates from bacteriophages, but it is carried by, among other strains, the E. coli BL21(DE3) strain. T7 RNA polymerase is located on the chromosome of E.coli BL21 (DE3) and is under control of the lacUV5 gene, which may be induced by lactose or IPTG. As IPTG is not metabolized by E. coli, while lactose is, IPTG was our inducer of choice [8].
The expression host can be cultivated and, in the case of the pET-11a system, induced to start expression of the recombinant protein. Growth of the culture can be tracked with absorbance measurements of optical density (OD). The protein expression is studied using SDS-PAGE.
Protocols
First curli detection protocol(purification of curli from E. coli MC4100)
Curli detection with sonication probe
Quick start Bradford protein assay
E. coli TG1, E.coli XL1-Blue and E. coli BL21(DE3)
Note: In cycle 1 and 2 of the project, the recommendations for a low copy plasmid were used, while during cycle 3, the recommendations for the high copy plasmid were used.
DNA purification from PCR mixture and gel
The protocol from Nucleospin Gel and PCR clean-up kit was followed for DNA purification.
Note: For work with pTRKH3-ermGFP use plates infused with 150µg/ml erythromycin, for work with pET11a, use plates infused with 100µg/ml of ampicillin. The competent cells were thawed and a small amount was added to the plasmid followed by gentle shaking.
References
[1] 1. Biesecker S, Nicastro L, Wilson R, Tükel Ç. The Functional Amyloid Curli Protects Escherichia coli against Complement-Mediated Bactericidal Activity. Biomolecules. 2018;8(1):5.
[2] Groenning, M. Binding mode of Thioflavin T and other molecular probes in the context of amyloid fibrils—current status. Journal of chemical biology, 2010;3(1), 1-18.
[3] Sabaté R, Saupe S. Thioflavin T fluorescence anisotropy: An alternative technique for the study of amyloid aggregation. Biochemical and Biophysical Research Communications. 2007;360(1):135-138.
[4] Xue C, Lin T, Chang D, Guo Z. Thioflavin T as an amyloid dye: fibril quantification, optimal concentration and effect on aggregation. Royal Society Open Science. 2017;4(1):160696.
[5] Zhang L, Li Z, Chen Z. Live cell fluorescent stain of bacterial curli and biofilm through supramolecular recognition between bromophenol blue and CsgA. Chemical Communications. 2020;56(37):5014-5017.
[6] Lizier M, Sarra P, Cauda R, Lucchini F. Comparison of expression vectors in Lactobacillus reuteri strains. FEMS Microbiology Letters. 2010;308(1):8-15.
[7] Part:BBa K1033225 - parts.igem.org [Internet]. Parts.igem.org. 2021 [cited 11 October 2021]. Available from: http://parts.igem.org/Part:BBa_K1033225
[8] TB055 pET System Manual [Internet]. Merck; 2011 [cited 13 October 2021]. Available from: https://www.merckmillipore.com/SE/en/product/pET-11a-DNA-Novagen,EMD_BIO-69436#documentation