We prepared the antibiotic stock solutions, the IPTG stock solutions and made LB medium. We accidentally made a solution of 25 mg/mL spectinomycin instead of 50 mg/mL, which means that we have to add double the amount of spectinomycin in the future (e.g. 200 µL instead of 100 µL for large cultures). Furthermore, we prepared medium for agar plates by autoclaving LB medium containing agar-agar.
The ordered ARG, TtrS and TtrR plasmids were delivered in dry swaps in DH5 alpha cells and were used to make small cultures for Miniprep preparation. After overnight incubation, we isolated the plasmids from the DH5 alpha cells with the Qiagen Miniprep kit and co-transformed the TtrR and TtrS plasmids into E. coli BL21 (DE3) cells and transformed the ARG plasmid into different BL21 (DE3) cells.
Furthermore, we made small culture glycerol stocks from the small cultures and stored them in the -80 °C freezer. In addition, more agar plates were poured containing only kanamycin and plates with spectinomycin and chloramphenicol. Unfortunately, the co-transformation of TtrS/R did not work, since the Drigalski spatula was likely too hot during the spreading. Therefore we executed the co-transformation again. However, the second co-transformation did not result in colonies on the plates either. Therefore, we asked our supervisor Yan Ni for advice, and she recommended using SOC medium instead of LB medium during the incubation of the transformed cells. We repeated the co-transformation with the SOC medium and had colonies on the plates. The transformation with ARG did work the first time.
This week we had planned to do the first ultrasound measurements, so we started with a small culture of ARG, made a large culture out of a prepared small culture and prepared the first phantoms containing the large cultures. During the measurement of the phantoms using the ultrasound, we could see a lot of white stripes on the bottom of the ultrasound images. Furthermore, we wanted to purify the ARG proteins to see if the cells did make the right proteins. However, due to a mistake in the protocol, we discarded our proteins and the protocol was not continued.
Additionally, we wanted to measure the GFP fluorescence of TtrS/R with different concentrations of inducers (to establish a dose-response) and therefore made a small culture and large culture. However, the LB medium that we used for the small cultures turned out to be contaminated and therefore no results were obtained. Furthermore, the tetrathionate inducer that we used should have been stored in the fridge. However, other researchers stored it at room temperature (for over a year), which likely resulted in the degradation of the tetrathionate. We noticed that it did not dissolve well enough in water and had a different color than was expected. Therefore, a new bottle of tetrathionate was ordered.
Since the ultrasound images of last week were not clear and showed white stripes, we decided to discuss these results with an expert in ultrasound imaging: Hein de Hoop. We prepared phantoms with only PBS agar agar and one also mixed with LB medium. With his tips, we continued with a small culture, large culture and the ARG measurement. Since we only saved the pictures instead of the raw data, image processing was not possible. Therefore, we could not see the differences between the images before and after collapse. Furthermore, we purified the ARG proteins and measured their concentrations using the Nanodrop. More on the improvements we made in the ultrasound measurements with the help of Hein can be found on the Engineering Succes page segment Ultrasound.
Since most members of our team were on holiday, we did not perform lab work this week.
This week, we wanted to repeat the TtrS/R GFP measurement again with new ordered tetrathionate and new LB medium. Therefore, we did a co-transformation with TtrR and TtrS plasmids using SOC medium. Afterward, we did small culture and large culture and measured the GFP and mCherry fluorescence signals with the plate reader and the photospectrometer. The plate reader and the photospectrometer results showed the same fluorescence intensity for all samples and the blanco.
Since our ARG plasmid was not Biobrick RFC or RFC compatible, we had to insert 3 mutations in the DNA. Therefore, we did site-directed mutagenesis with our plasmid and made a small culture of the colonies for sequencing. Unfortunately, the sequencing results contained multiple non determined bases, which made the results not useful. Next to site-directed mutagenesis, we did a transformation and small culture of wildtype ARG and a large culture for ARG ultrasound imaging. During the ultrasound imaging, we received help from Hans-Martin Schwab, for more information on this see Engineering Succes. He recommended using a different protocol, to reduce air bubbles, for making these phantoms by using a heating plate instead of the microwave for heating and mixing the agar agar with the PBS. We also used this protocol and made new ultrasound images. Furthermore, we purified the wildtype ARG proteins and made an SDS-PAGE. Since the SDS-PAGE was still too blue after shortly washing, we washed over the weekend and stained and washed again on Monday. Furthermore, the bands in the gel were not bright because the concentration of the proteins was too low.
This week, we made a picture of the SDS-page gel from last week and we repeated the TtrS/R GFP measurements, however, this time using different concentrations of dox, IPTG and tetrathionate, instead of a tetrathionate gradient. We made cultures and performed the GFP measurements again with the plate reader and the photospectrometer. The plate reader results gave similar fluorescent intensity for all samples and blanco. In contrast, when measuring the emission at different wavelengths at an excitation of 485 nm, the photospectrometer gave the expected peak with it’s maximum at the expected wavelengths. Furthermore, there a different fluorescence intensity was observed for the samples and the blank measurement and also different intensities between the samples were observed, as predicted. Therefore, further experiments were conducted using the photospectrometer.
We also amplified the amount of mutant ARG plasmid to obtain a higher concentration of mutant ARG for transformations. Lastly, we tried synthesising the design A plasmid, containing insert A, by restriction and ligation. However, we did not properly calculate the right amount of insert needed and therefore, the gel electrophoresis showed no bands and a smurred ladder. We choose not to continue with ligation and we first planned a meeting with expert Alexander Gräwe. More about the information we received from Alexander can be read on the Human Practices) page.
This week we measured the TtrS/R GFP intensity using a dox gradient. We performed the measurements for GFP and mCherry and the results are shown on the Results page. Furthermore, we successfully performed restriction and ligation of design A with insert A and tried to perform the co-transformation with the TtrS, TtrR and design A plasmids into BL21 (DE3) cells. Unfortunately, after co-transformation in BL21 (DE3) the agar plates showed no colonies. After meeting with Alexander Gräwe, it was concluded that the concentration of the design A plasmid was too low.
This week, we repeated wild type ARG ultrasound measurements, however, this time using a different gradient of IPTG concentrations than used in week 32. The wildtype ARG was purified again and placed on an SDS-PAGE gel and ultrasound measurements were performed. The ultrasound images were different from last time, which forced us to do some trouble shooting. Since the OD600 of our large cultures at the moment of induction was lower than the previous time (0.6 instead of 2), the phantoms contained not enough cells and thus the ultrasound images showed no difference between before and after collapse of the gas vesicles (except the concentration of 1000 µM IPTG). For the phantoms containing 10.000 µM IPTG, we suspect that the IPTG concentration was too high and the cells were killed, due to the toxic character of IPTG . Moreover, the design A plasmid was amplified with a small culture and Miniprep was performed to obtain a higher concentration of plasmid. The previously executed co-transformation unfortunately did not work, so we repeated the experiment. However, this co-transformation also did not work, while the positive control showed colonies.
Besides, we performed TtrS/R GFP measurements again with a control and samples with 3 different dox concentrations with and without tetrathionate. The results can be seen on the Results page.
Since we wanted to produce the full dose-response curve for the TtrS/R system, we repeated the very first experiment for TtrS/R, however, this time the optimum concentration of dox (0 ng/mL) was used. The thought behind this choice is elaborated on the Engineering Success page. Unfortunately, we discovered that the LB medium used for the small cultures was contaminated. There was still some dose-response visible in the range as expected, but nothing could be concluded from these results, since all three controls failed.
Additionally, we tried the co-transformation of design A again by first making our TtrS/R cells competent. These cells already contain the TtrR and TtrS plasmids and the design A plasmid could be transformed into these competent cells. The co-transformation plates did not show any colonies. Furthermore, we started with preparations for part improvement by preparing agar plates and by transforming the wildtype pSB1C3-RFP coding device from the distribution kit. We made a small culture of the transformed cells and isolated the DNA using Miniprep.
This week, we tried the co-transformation of design A again with the TtrS/R competent cells. Since this did not work, we transferred to our design B approach and did the restriction and ligation of the design B plasmid. After ligation, we transformed the design B plasmid into Novablue cells. Unfortunately, this did not work so we tried the transformation again using XL10-Gold and NovaBlue cells. However, we still did not see any colonies on our plates. In addition, we tried the transformation of our mutant ARG again in BL21 (DE3) cells, which did not work.
Furthermore, for part improvement, we amplified the ordered 5’UTR insert using PCR and purified the DNA. Afterward, we restricted and ligated the pSB1C3 plasmid from the iGEM distribution kit with our 5’UTR insert and transformed them into BL21 (DE3) and DH5 alpha cells. We also transformed the wildtype pSB1C3-RFP coding device into BL21 (DE3). Since only colonies of the DH5 alpha cells were visible on the plates, we made a small culture of them and isolated the DNA with Miniprep. Furthermore, we transformed the wildtype pSB1C3-RFP coding device again in BL21 (DE3) and also the isolated ligated pSB1C3 in BL21 (DE3).
This week, we continued with part improvement by amplifying the ligation product and the wildtype plasmid and continued with isolating the DNA using Miniprep. These plasmids were transformed into BL21 (DE3) cells, however, even after a few attempts, there were no colonies present on the agar plates, while the positive control was overgrown. To check the ligation product and the wildtype pSB1C3-RFP coding device, we sent these plasmids for sequencing. Furthermore, the restricted wildtype ARG and PCR product of the insert from design B were also sent for sequencing to check if the restriction sites were cut and if the PCR reaction worked.
Since the previous dose-response curve attempts failed, we repeated this experiment again with the final concentrations of inducers and a tetrathionate gradient, as can be seen in the Results. Alongside this experiment, we tried the restriction and ligation of design B again and transformed the ligation product into XL10-Gold and TOP10 cells. To achieve this, the insert B first had to be amplified by PCR. For part improvement, we amplified the 5’UTR insert with PCR and transformed the pSB1C3-RFP coding device plasmid into DH5 alpha and TOP10 cells.
This week, we repeated the restriction/ligation of design B plasmid for the last time, with newly prepared insert and plasmid, and transformed the ligation product into XL10-Gold and TOP10 cells. Since colonies appeared on all plates except the negative control, we made a small culture of the TOP10 and XL10-Gold colonies with the ligation product and isolated the DNA. We co-transformed the ligation product together with TtrS in XL10-Gold, TOP10 and BL21(DE3) cells which resulted in multiple plates with colonies and thus successfully transformed design B. Furthermore, we tried to co-transform design A plasmid again in BL21 (DE3) cells, which as expected did not grow.
In addition, we tried the restriction/ligation again for pSB1C3-5'UTR and successfully transformed the ligation product into TOP10 and DH5 alpha cells. We made a small culture and large culture, and measured the RFP fluorescence.
However, the laboratory we worked in suffered from a cleaning problem, which means that our large cultures were probably contaminated and therefore, the large cultures made this week have no reliable results. The ultrasound images had a lower intensity compared to the previous images, which caused the filters to fail. Therefore, a different filter was used, however, this filter influenced the results. Furthermore, no significant difference between induced and not induced images could be seen. The same problem can be seen with the RFP fluorescence measurements. Therefore, these results will be excluded. Lastly, we sent our ARG insert A and B together with pSB1C3-5'UTR for sequencing.
Dvorak P, Chrast L, Nikel P, Fedr R, Soucek K, Sedlackova M et al. Exacerbation of substrate toxicity by IPTG in Escherichia coli BL21(DE3) carrying a synthetic metabolic pathway. Microbial Cell Factories [Internet]. 2015;14(1).