Team:Rotterdam HR/Results
iGEM Rotterdam HR 2019
Results
gBLOCKs
The gBLOCKs coding for fusion proteins of N-/C-TEV with the zinc finger arrays was designed on www.zincfingertools.com as previously described in our Drylab page.
Sense and antisense strands (IDT) were annealed and analyzed on 0.8% agarose gel (Figure 1).
Figure 1: gBLOCKs 1 and 2 on 0,8% agarose gel. 2-Log DNA ladder of New England Biolabs (NEB) is used as marker.
The pSB1T3 and pSB1A3 plasmids gave the two bands that came close to our expectation after digestion with the restriction enzymes EcoRI and PstI (figure 2).
Figure 2: pSB1T3, pSB1A3 and gBLOCK2 on 0,8% agarose gel. 2-log DNA ladder (NEB) is used as marker. * Digested
Unfortunately, the ligation of pSB1A3 or pSB1T3 with gBLOCK2 did not work because all the colonies (and the negative control) were pink. The pSB1T3 and pSB1A3 plasmids both contain the RFP coding device (BBa_J04450), this means it will have a pink color. When a BioBrick has been inserted into this plasmid, it will result in a white color because the RFP coding device will be cut out of the plasmid with the restriction enzymes. So, the pink colonies mean that no insert has been introduced into the iGEM plasmid (Figure 3) and thereby we think that the ligation of our gBLOCK into the plasmid did not happen. As negative control only the dephosphorylated plasmid (pSB1A3 or pSB1T3) has been used, so without the gBLOCK2.
Figure 3: Results of the transformation of pSB1A3 with gBLOCK2 on the left. On the right the negative control.
After redoing the experiment multiple times, we only got “empty” agar plates. This was not due to the transformations, because within the same experiment we also transformed a pUC35 plasmid and this resulted in multiple colonies on the agar plate. So, probably there went something wrong with the ligation step. For the ligation, different kits were used to be sure that the materials of the kit worked, these kits gave the same results. The pUC35 plasmid was digested with a different restriction enzyme compared to the pSB1A3 plasmid. Therefore, it is possible that something went wrong with the digestions.
Before we pursued further experimentation, we focused on amplifying our DNA of gBLOCK2 by PCR. After many experiments, the DNA concentration of gBLOCK2 was not enough to proceed. The PCR gel extraction resulted in low DNA concentrations, these were too low to use it for further experiments. Therefore the focus was on other experiments instead.
DNA bridges
Unfortunately, our DNA bridges are being held at Customs in Rotterdam. Therefore we also didn’t work with aptamers because we first wanted to know the ideal distance and orientation for the TEV proteins and if the rest of the system could work. We did not want to waste our resources and rather focused on other parts of the system.
Update
After we came back from Boston the DNA bridges finally arrived!
TEV-cleaving site – β-Lactamase
TEV will only “cleave off” β-Lactamase (bla) from its inhibitor if there is a TEV-cleaving site. To find an inhibitor protein, multiple (random) proteins had to be tested. For this reason, bla coding constructs with N-terminal or C-terminal TEV-cleaving sites were created after performing a fusion PCR. For the fusion PCR pUC18 has been used as a template. Different fragments with bla and TEV were made by using different primers as described in the Protocols page. The last primers that are used for the fusion PCR contain the RFC10 prefix or suffix site.
Figure 4: Fusion product of the TEV-cleaving site with bla on 0,8% agarose gel.
After performing the fusion PCR, a product with the TEV-cleaving site attached to bla had been made which is shown in Figure 4. This product and the plasmid pSB1C3 were cut with restriction enzymes XbaI and SpeI. This resulted in multiple-bands on the gel that were not expected, we concluded that the plasmid pSB1C3 wasn't a suitable plasmid to use.
Because restriction enzymes tend not to cut well near the end of PCR products, the option was to clone the TEV-cleaving site-bla product by blunt-end ligation into a cloning vector. The pUC35 is a pUC18 derived plasmid with a chloramphenicol resistance marker instead of the ampicillin resistance marker. This plasmid was constructed by other students of the Rotterdam University of Applied Sciences. The TEV-cleaving site-bla product was ligated into the pUC35 plasmid that was digested with SmaI-HF. After transformation and blue-white screening, the plasmid DNA of the white colonies were isolated and sent for sequencing. From the sequence results, we concluded that no TEV-cleaving site-bla fusion constructs had been obtained.
TEV
We have fusion proteins in the isolated form of GST-TEVcleavingsite-BRCA2peptide (GST-TEV-BRCA2) and functional TEV proteins. This combination could serve as a positive control for experiments on TEV-cleaving site-bla fusion constructs. We made a mix of the two proteins and incubated them on 37 ⁰C. Samples were taken 1 hour, 2 hours and 4 hours after incubation initiation. This was done in duplicate. In lanes 1-4 and 6-7 (Figure 5) GST-TEV-BRCA2 is visible which has been incubated with the TEV protein. A split in the protein is seen, however, the TEV protein itself is not visible on the gel. Lanes 8 and 9 should show only TEV protein, but no bands that indicate presence of TEV are visible.
The same experiment was repeated, but however, reproducing the experiment gave different distinct results. In one of the results, the fusion proteins were undigested and remained far above in the gel, and TEV was again invisible. In order to avoid doing experiments with samples that may not contain the TEV protein at all, we recommend HPLC analysis for future experimenting. This will result in more specific detection of the presence of TEV proteins.
Figure 5: Testing TEV with GST-TEV-BRCA2 on 10% SDS-PAGE gel. Blue Prestained Protein Standard(NEB) is used as marker.
Nitrocefin
Figure 6: Nitrocefin test of a bacteria with ampicillin resistance. On the top you can see the negative control, on the bottom the two test samples are shown.
A one-month-old bacteria with ampicillin resistance has been used to look if the nitrocefin shows a color change in the presence of β-Lactamase. The bacteria have been resuspended in phosphate buffer, so as negative control phosphate buffer was used. After 8 minutes the color changed from yellow to red. Nitrocefin could be used in the lab for testing the system, but nitrocefin is less safe for our final product. So, a replacement is needed.
Chromoproteins
One of the criteria for bronze is to perform characterization tests on parts from the registry. Our choice was to characterize the absorbance spectra of the chromoproteins made by the iGEM team Uppsala in 2013. An addition to the characterization tests is to see whether there will be a change in the absorbance spectrum when the proteins are suspended in acidic or basic environments.
For the first experiment, a full-spectrum analysis(300-1000 nm) of fresh lysate chromoproteins purple (BBa_K1033917) and pink (BBa_K1033926) was performed. Results can be seen in Figures 8-11.
The second characterization experiment (figures 13-16) was to test the effect of pH: 0.3, 4.3, 7.3, 10.3 and 14. The tests have been performed twice in different wells of a 96-wells plate, 100 uL of each chromoprotein sample was mixed with 100 uL of different pH-samples.
Figure 7: Absorbance spectrum of saline.
Figure 8: Full spectrum analysis of asPink.
Figure 9: Duplicate of the full spectrum analysis of asPink.
Figure 10: Full spectrum analysis of gfasPurple.
Figure 11: Duplicate of the full spectrum analysis of gfasPurple.
For the different pH experiments, the results are shown below.
Figure 12: Full spectrum pH-analysis of asPink.
Figure 13: Duplicate of the full spectrum pH-analysis of asPink.
Figure 14: Full spectrum pH-analysis of gfasPurple.
Figure 15: Duplicate of the full spectrum pH-analysis of gfasPurple.
In conclusion, increasing the pH of the suspension sample shifts the absorbance spectrum of both chromoproteins to the left starting from pH7.3 (natural conditions in the cell), to decreasing wavelength.
