Team:TU-Eindhoven/AnearobicTesting

Abstract

Anaerobic protein expression is wanted for protein expression when bacteria have targeted the tumour (hypoxic environment). The proteins that were chosen for this experiment will be induced by triggering a FNR promoter, which is triggered under hypoxic conditions. The proteins that have been chosen are arginine and lysine rich, which give the expressed proteins the feature of generating CEST contrast which can be measured with MRI machines, making bacterial protein expression a possible tumour targeting mechanism. Testing the promoter under hypoxic conditions turned out that no proteins were expressed in BL21 bacteria, but EGFP was produced in XL-1 Blue and in MG1655 strains. XL1-Blue strains appear to be the best strains for anaerobically induction of CEST protein expression, since there is an increase in intensity of EGFP.

Introduction

Within the project of tumour targeting with bacteria, a promoter has to be tested on certain conditions in which tumour cells live. Near and in tumours there is a hypoxic environment (lack of oxygen). This means that the bacteria needs to express their proteins under hypoxic conditions, when tumour targeting is wanted. The proteins that will be expressed are arginine and lysine rich proteins, which can give the proteins the feature to generate CEST contrast which can be measured with MRI machines. For a proof of concept, the promoter that has been chosen (an FNR promoter, since it is an oxygen concentration regulated transcription factor, which will induce transcription when the environment has low oxygen concentrations to turn the metabolic activity in bacteria from aerobic to anaerobic metabolism), has to be tested under hypoxic conditions.

Construct Design

The construct was designed as shown below. A promoter was needed, which is explained in more detail below (FNR). For purification of the proteins, a His-Tag was included in front of the protein and a Thrombine cleavage site for cutting off the His-Tag after purification. Since the His-Tag will not be removed, it is useless, but to keep the differences between the anaerobic and aerobic sequences as small as possible, a Thrombine cleavage site was inserted. Then the sequence for the CEST proteins/EGFP. A spacer sequence and a terminator were placed behind the protein sequence. For more information about the construct design, see Construct Design and Chasis.

FNR

Since anaerobic expression is desired, a FNR promoter was inserted in front of the sequence for the desired protein. The promoter design was taken from research by Barnard et al. [1] For more information about promoter design, see [2013.igem.org/Team:TU-Eindhoven/FNRPromotor]. The chosen promoter had 1.8 times higher activity than the most commonly used single binding site FNR promoter. The FNR promoter is regulated by fumurate and nitrate reduction (FNR). This is a transcription activater, that can control the expression of genes when oxygen concentrations are low. The active state has a Fe-S group and when it is bound to [4Fe-F2]2+ clusters, it can form dimers. The dimers are the active transcription factors. Oxygen can destabilize the dimer-clusters which results in an inactive state of the transcription factor. For more information about the FNR dynamics, see [2013.igem.org/Team:TU-Eindhoven/ODEModel].

Methods and Results

Cloning

Once the pUC57(s) constructs were delivered, we could transform them into NB bacteria for amplification of the vectors. When they were amplificated, a digestion on the constructs as well as on the pBR322 vector is performed with EcoRI and HindIII restriction enzymes to cut out the whole sequence (so with the promoter until the terminator) out of the pUC57(s) construct. Chosen was for pBR322 since it has no promoter in it which is helpful so that we could test the FNR promoter. Once the pUC57(s) constructs as well as pBR322 vector were digested a gel extraction was performed to cut out the desired bands. Then a ligation is performed to get the protein sequence from the pUC57(s) constructs into the pBR322 vector. After ligation the constructs were plated with ampicilin antibiotics and grown overnight to create colonies. To check whether ligation was succeeded a colony PCR procedure was performed and the results were loaded on an agarose gel.

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Then from the chosen constructs (Pro1(1,2), 1PJN(1,2), 1ETF(3,4), P(RG)(1,2), P(RS)(1,5), EGFP(1,2), 1G70(3,5), P(TK)(2,3), P(KS)(1,3) since these bands seem to be correct since they are at the expected hight), small cultures were set in, again with ampicilin, and grown overnight. To retain the vectors, a miniprep procedure was performed and once the constructs were sequenced and seemed to be correct. Then the ligated vectors were transformed onto BL21 bacteria for protein expression.

Protein Expression

For anaerobic protein expression a Biofermentor, wherein anaerobic conditions can be reached by creating a flow of nitrogen through the culture, was used. First the ligated vectors were transformed onto BL21 bacteria by heat shocking the bacteria. To test the FNR promoter the pBR322-EGFP construct was used, since it can be measured very easily. 4L of LB medium was made and autoclaved within the vessel. Then the bacteria were added and were grown aerobically until the optical density was about 0.6. Once the optical density was about 0.6 the air supply was turned off and only nitrogen was blown through the culture. At several moments a sample was taken for the measurement of fluorescence. The culture was held the whole experiment at 37 °C (body temperature).

It turned out that no EGFP was measured, indicating that the promoter did not work as expected. A possible explanation for this problem was a mutation in the FNR coding gene within BL21 bacteria. This would mean that FNR production would not occur whilst the proteins are being expressed in BL21.

Since protein expression in BL21 bacteria was not succeeded, the pBR322-EGFP vector was then transformed, by heat shocking, into XL-1 Blue bacteria and by electroporation into MG1655 strain which was provided by the University of Groningen. The same procedure for the anaerobic protein expression as was executed for the BL21 was now performed for the XL1-Blue and the MG1655 strain. In Figure 4 and 5 the gel analyses of the bugbustered samples from the anaerobic induced EGFP expression is shown. After His-Tag purification of the samples, another 12% SDS gel analysis was performed. From the XL1-Blue samples the hole sequence that was obtained from the Biofermentor was purified (since the gel seemed promising, because of the increase of intensity of certain bands, though it is at the wrong height (around 40 and 45 kDa). There is a less clear band at the height about 29 kDa, which could indicate that EGFP was expressed. For the MG1655 samples only samples 1, 8 and 9 were taken to see whether there was an increase in intensity (this gel was less promising than the XL1-Blue samples).

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The gel with the purified samples is shown in Figure 6. For the XL1-Blue samples there is a band around 50 kDa that increases in intensity. There is a little band that might increase in intensity at around 30 kDa. This could indicate that EGFP at the right weight is expressed. Since concentrations after purification were very low, it can be that the intensity is not that high. For the MG1655 a clear band is located around 45 kDa, which seems to correspond with the bonds seen before on the unpurified gel, but it does not give us any information about EGFP expression.

Fluorescence Measurements

From the purified samples also samples were taken for fluorescence analysis. EGFP emission spectra were desired to have an increased intensity when the samples were taken at later points in time. The samples from MG1655 did show the right emission spectra, but there was no increase of intensity at all. Scattering was excluded since a shift in excitation wavelength did not result in a shift in emission spectra. The samples of the XL1-Blue expression, did show an increase in intensity. The results are shown in figure 6. Here scattering was also excluded. And the values of the peaks were shown in figure 8. A comment on these measurements is that the gain was high (187), which indicates that very low concentrations are present.

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In figure 8, the second sample shows a decrease in intensity. This can be caused by the switch from aerobic to anaerobic metabolism so that the EGFP that was produced can be degraded until the bacteria are fully switched to anaerobic metabolism. Then the peaks are getting higher, which can be caused by higher concentrations EGFP that was anaerobically produced by the bacteria. Another reason for this can be that the bacteria did still grow and that the produced EGFP is background expression. The intensity of the last sample was decreased with respect to the peak around 18-19 hours. This can also be caused by degradation of the produced proteins.

Discussion and Conclusions

SDS gel analysis of the unpurified samples from the XL1-Blue culture indicate that there are several protein that get more expressed when the samples were longer under anaerobic conditions. Here the bands of interest were at the height of 45-50 kDa and around 30 kDa. After purifying by the His-Tag, the SDS gel did not contain that clear bands as it had on the unpurified gel, but there are bands at the same height (around 45-50 kDa) and bands around 29 kDa, which have no high intensity, but does show a little increase in intensity. Fluorescence measurements of the purified samples indicate that there is indeed EGFP expression and that the intensity gets higher when the cultures were longer exposed to the anaerobic environment. Here we have to place the comment that there was a maximum in the intensity. The last measurement was lower than that one of 10 hours before the end of the experiment. This can be caused by degradation of the proteins. Since this gap between the two last measurements was caused by overnight experiment, we can give no conclusions about this event. Scattering was excluded by taking other excitation wavelengths that resulted in the same emission spectra. We can conclude that EGFP is expressed, though it were very low concentrations which resulted in low intensity bands on the purified SDS gel and a high gain for the fluorescence measurements.

For the SDS gel of the unpurified MG1655 samples we saw some bands that indicate an increase of protein expression. These bands also show up on the purified samples SDS gel. The bands are at the same height as the bands on the unpurified gels, so they correspond. The fluorescence measurements for the purified MG1655 samples showed the correct emission spectra for EGFP. The spectra for the samples that were longer exposed did not have an increased intensity. Scattering was excluded by taking other excitation wavelengths, which resulted in the same emission spectra.

Overall we can conclude that the FNR promoter does work and EGFP is produced anaerobically.