Difference between revisions of "Part:BBa K1123005"

Latest revision as of 18:05, 17 October 2018

EGFP Protein Production Construct

This composite part has been built up from a number of separate BioBricks, the most prominent of which the FNR promotor and the EGFP Protein sequence. The FNR promotor sequence enables us to bring the EGFP protein to expression anaerobically. Almost all E.coli strains posses a form of this promotor to induce metabolic processes once the bacteria enters anaerobic regions. The EGFP protein is a frequently used fluorescent protein which we are expressing with the sole purpose of testing the protein expression under the FNR promotor.

TU-Eindhoven Sequencing Results

This construct has been submitted to the iGEM registry and has been privately sequenced by the iGEM 2013 TU-Eindhoven. The sequencing results were positive indicating that the sequence and part submitted to the registry are correct. Sequencing was performed by BaseClear Netherlands

Sequence and Features

Characterization

This part shares general concepts with the Valencia 2012 part: BBa_K763002

Characterization by the TU Eindhoven 2013 iGEM Team

As part of the iGEM 2013 competition the TU-Eindhoven team attempted to create a promoter which would induce protein expression under anaerobic conditions. This functional promoter could then be used to express CEST proteins (which should enable MRI imaging) upon the bacterium entering tumour regions. To test the functionality of the promoter we decided to express EGFP under anaerobic conditions. EGFP can be easily analysed making it easy for us to characterize the workings of the FNR promoter. This expression of EGFP was performed three times, the first two both at 0% Oxygen whereas the third time we attempted to express at 5% Oxygen.

If we manage to express EGFP using this FNR promoter: BBa_K1123000 then we will be able to express other CEST proteins ( BBa_K1123001, BBa_K1123002, BBa_K1123003, BBa_K1123004, BBa_K1123006, BBa_K1123007, BBa_K1123008, BBa_K1123009) anaerobically with a high probability of success.

For the EGFP expression under influence of the FNR promoter we cloned this biobrick into a pBR322 vector. This vector was then transformed into BL21 bacteria. Colonies of this transformation product were grown in (6) 8mL culture tubes and were then injected into a 4L LB solution, found in a bioreactor. After the optical density had reached 0.600 the chamber was constantly flooded with Nitrogen for over 24 hours, having samples taken every few hours during the day. After this anaerobic expression was complete the samples were analysed but no EGFP could be observed. A disappointing result. Upon closer investigation of the BL21 E.coli strain, we found that the FNR production, necessary for the proper functioning of the FNR promoter was absent.

In response to this lack of FNR within the BL21 bacteria it was decided that we attempt expression within a second E.coli strain. For this the XL-1-Blue strain was chosen. Before performing the actual experiments of course some research was performed to ensure that our assumptions were also true.

Once certain this strain was FNR compatible we transformed this biobrick, once again in the pBR322 vector, into XL-1-Blue E.coli bacteria. These were grown on agar and then picked, placed in 8mL of LB and then brought over into 4L of LB medium. Here they were allowed to grow aerobically until the Optical Density (O.D.) was 0.600 or higher. This protocol was retained for all three expression experiments.

Anaerobic expression at 0% Oxygen saturation.

Anaerobic expression at 0% Oxygen saturation.

Anaerobic expression at 5% Oxygen saturation.

The graphs above show the growth curves of the bacteria during the course of the experiments. During the first experiment the optical density was measured up until the oxygen was removed from the vessel containing the bacterial culture medium, during the second and third experiments the optical density was measured continually throughout the experiments. In all three experiments, upon reaching an optical density of approximately 0.600 the oxygen inlet was closed and the vessel was flooded with nitrogen until the desired oxygen concentration was reached. The medium was then continually regulated to maintain this desired concentration. For all three experiments you can see that cells are still in their exponential growth phase when the oxygen concentration is reduced. Throughout the rest of each of the experiments, samples of the culture medium were taken at regular intervals so that we could follow the production of EGFP as a result of the anaerobic environment which sets in motion the DNA transcription using FNR promoter.

During the experiments the vessel was left at 37°C for approximately 25 hours before the last samples were taken. At this point all samples taken up until that point were spun down and pelleted so that the bugbuster protocol could be performed. As the samples we had taken whilst performing the expression were no larger than 25mL the volumes we had to work with were relatively small. After performing the bugbuster protocol the solutions were poured into small eppendorf tubes and spun down. The product hereof was a supernatant containing our EGFP protein and a small pellet of waste.

For the first experiment only a small portion of this supernatant was then taken and loaded onto a 12% SDS gel.

Here the time in minutes before each sample was taken is as follows: t1 = 0 minutes, t2 = 39 minutes , t3 = 180 minutes, t4 = 375, t5 = 1190, t6 = 1710.

At first we were pleasantly optimistic about the results, as you can see there is an obvious increase of a certain protein in accordance with time increase. However, under scrutiny, it becomes visible that the protein which is being expressed so clearly has a molecular weigh far higher than that of EGFP which we expect at approximately 30kDa. The red boundary shows which bands on the gel we believed could be our EGFP protein.

To improve upon this result, we purified the supernatant. This was also done for the supernatants obtained during the second and third experiments. We made use of small His-Tag binding spin columns. These would bind only to our EGFP protein, which should be bound to the His-tag we attached. After purifying the same samples were loaded onto 12% SDS gels which can be viewed below.

Gel show the same 6 samples as shown in the previous unpurified gel.

Here the columns marked C- and C+ are respectively a negative and positive control. The negative control consists of a sample taken from a medium which was allowed to grow aerobically for 25 hours. The positive control is a known EGFP samples. Its height can be put down to its binding with a CNA35 protein construct. Futhermore two sets of 6 samples can be seen. The first 6 time samples were taken during experiment 2 (at 0% oxygen concentration) and the second set of 6 were taken during experiment 3 (at 5% oxygen concentration)

Due to the purification we did of course dilute the protein concentration somewhat, and the low starting concentration of EGFP as seen on the first gel of the first experiment, it was not entirely unrealistic for us to only see such a slight band on the SDS gel. This is also in accordance with the experiment as we were not over expressing the EGFP protein but expressing it using a standard metabolic promoter. We do however see the thick band reappearing in each of the three purified gels, although with a much lower intensity for the first experiment compared to the second and third experiment.

As we wished to show that the light band really does correlate to EGFP, we performed a western blot on a secondary gel which was equal to the purified sample gel for experiments 2 and 3. A secondary gel was used as a western blot cannot be performed on a gel which has been stained with coomaaise, as ours were. During the western blot, we made use of GFP binding antibodies to selectively bind only our EGFP protein.

Western blot of purified EGFP samples for experiments 2 (at 0% Oxygen0 and 3 (at 5% Oxygen). The samples here are loaded equally to the samples loaded for the SDS gel.

On the image above we clearly see that EGFP really is the lower band we keep seeing on our SDS gels. What the upper thick band relates to is unknown, but the fact that it does not bind to EGFP binding antibodies tells us that it is not related to EGFP in any way.

One final test performed to check whether or not EGFP was being produced came from a fluorescence measurement. Herein we tested the emission spectra of each of the samples by pipetting a portion of the purified sample into a wells plate. Graphs showing the emission intensities for each of the samples are given below..

EGFP intensities over time when expressed at 0% Oxygen

EGFP intensities over time when expressed at 0% Oxygen

EGFP intensities over time when expressed at 5% Oxygen

In each of the three graphs we see that the intensity of EGFP emission increases in accordance with increasing time steps. However we also see that these intensities begin to decrease if left too long. This is in accordance with the intensity of the bands seen on both the purified SDS gels as well as the western blot. As each of the three graphs differ from one another not concrete conclusions can be drawn, however we expect that the highest concentration of EGFP will be found somewhere around 12-14 hours into expression. It should be noted that the intensities of EGFP retained from experiments 2 and 3 have been normalized with their respective Optical Densities, meaning that these graphs give a slightly better indication of the actual EGFP production per bacterium.

With these results we can be quite conclusive in saying that there was EGFP expression in each of the three experiments, induced by the hypoxic conditions in the medium over the course of time. We do however have to take in to account that the actual expression concentrations were extremely low. On the plus side the promoter does seem to be effective within anaerobic conditions.

Characterization by the Franconia 2017 iGEM Team

We further characterized the EGFP to supply a broader understanding of the properties of this fluorescent protein. We therefore measured the absorption and emission of EGFP. The regarding graph is given below (Figure 1).

Figure 1: Fluorescence spectrum of EGFP showing absorption and emission at variant wavelengths.

The results show a difference in the wavelength of the absorption and the emission. The absorption of EGFP has its maximum at a wavelength of 487.5 nm and the emission maximum lays at a wavelength of 508.3 nm. The overlap of absorption and emission can be seen in the area around 500 nm.

We further plotted the measured values of the excitation wavelength and the emission wavelength of EGFP to obtain a height profile given in the diagram below (Figure 2).

Figure 2: Excitation wavelength and emission wavelength of EGFP.

The diagram displays the distribution of the different wavelengths. The red colored area reveals the overlap of excitation and emission at its uppermost distinctness.

Characterization by the TJU_China 2018 IGEM team

We firstly amplified the eGFP fragment from BBa_K1123005(iGEM13_TU-Eindhoven) by PCR.

Figure 1. Amplification of eGFP fragment (BBa_K1123005) by PCR. Lane M, marker. Lane 1, 25ng eGFP fragment. Lane 2, 50ng. Lane 3, 100ng. Lane 4, 150ng. Lane 5, 200ng. Since this fragment contains the cleavage site of our sgRNA, we used it to test the in vitro cleavage activity of our CRISPR/Cas9 system. Please notice that our cleavage site was not so suitable for this fragment since it was designed for plasmid cutting(fragment of 720bp is cut into fragments of about 80bp and 640bp).

Figure 2. In vitro cleavage of eGFP fragment. Lane M, marker. Lane 1, eGFP fragment. Lane 2-3, sgRNA:Cas9:DNA=10:10:1. Lane 4-5, sgRNA:Cas9:DNA=10:20:1. Lane 6-7, sgRNA:Cas9:DNA=20:20:1.