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A | A | ||
<img src="https://static.igem.org/mediawiki/2015/e/ea/Warwick_sZF_10_oligo_uninduced.png"> | <img src="https://static.igem.org/mediawiki/2015/e/ea/Warwick_sZF_10_oligo_uninduced.png"> | ||
| − | |||
<br> | <br> | ||
<br>B | <br>B | ||
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Our original full construct containing Lpp_OmpA and Zif268 were cloned successfully into the pSB1C3 plasmid and transformed into chemically competent DH5α-Z1. Then we cloned INP, BclA, and sZFs 2,10 & 14 individually into our construct gene, using the restriction sites we included in the design. Results were confirmed by sequencing colonies picked from antibiotic selection plates.
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We showed that we could bind aminated DNA to slides using a fluorescein-labelled oligonucleotide. Two slides were used: a GOPTS-treated slide that should bind aminated DNA, and a control slide that was cleaned like the GOPTS slide, but was not treated with the GOPTS solution, and should not bind DNA (aminated or otherwise). Our experiment showed fluorescence only on the treated slide, showing that our DNA-immobilisation technique was valid.
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Our objective was to prove that the modified protein was being expressed on the surface of the plasmid transformed cells.
Upon comparison of image B and C,it is clear that the modified protein is being expressed upon induction with IPTG. However, the wild type cell shows fluorescence as well, suggesting that there is an error with our method of washing the cells after the fluorescent antibodies have been applied. The affected cells would fluoresce regardless of surface zinc finger expression.
Images A and B show no fluorescence or noise, however image C contains fluorescence, if it is very little. The background noise on image C is reduced due to the microscope's automatic contrast. This suggests that very few of our cells were expressing the protein. This could be due to incorrect growing or mounting of the cells.
From the images, only noise was detected on any of the slides. This could be caused by the protein not being expressed on the surface of the cell. A non-fluorescent image was taken of the induced slide to confirm this. This image shows that while there are clearly cells present, none of the cells are fluorescing. A random mutation in the colony that these cells were grown from could be the cause. While this plasmid was sequenced successfully, the colony that these cells originate from came from a replating of the original successful colony.
These induced cells are the most clearly fluorescent from the set that was measured. Based on the previous results we decided to alter the protocol for preparing cells for microscopy, instead of fixing the cells on a glass slide, these cells were in liquid medium on the slide. The alternative protocol can be found here. By comparing images A and C it is clear that the fluorescence is caused by the protein being expressed on the cell surface. Image B shows that the Pl_lac promoter did not leak and cause uninduced cells to express the protein in great enough numbers to be visible.
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When testing the binding of the fluorescently labelled oligonucleotides to the zinc finger proteins, we would expect to see fluorescence only when the correct oligonucleotides were added to the induced cells for each zinc finger. Instead, we found that all of our images showed fluorescence.
There could be many reasons for this, including background noise, errors in the washing method, and fluorescence due to the use of LB.
The exception to this was the experiment using sZF10. For these cells, we saw very little fluorescence (due to background noise or a leaky promoter) for the uninduced cells (A), but considerably more for the induced (B). However, due to the patterns in the image, we cannot assume that the fluorescence is a positive result for the expression of sZF10; it may just be background noise again.
A
B
