From 2014.igem.org

Latest revision as of 03:11, 18 October 2014

Fluorescent Protein Analysis

From our analysis of possible fluorescent protein reporters, we selected yellow fluorescent protein (YFP) and red fluorescent protein (RFP)

Construction: Overlap PCR

The estrogen responsive intein had 3 components, 1. the N-terminus of the S. cerevisiae VMA intein, 2. the human estrogen receptor ligand binding domain and 3. the C-terminus of the intein. The sequence was synthesized in two pieces as gBlocks from IDT, and the two pieces were put together using overlap PCR and clones were sequenced. A clone with the correct sequence was used as template for PCR with primers containing extensions that overlapped T7 RNA Polymerase (T7 RNAP). The intein was to be inserted between the 491 and 492 residues of the T7 RNAP so the N-terminus and C-terminus of T7 RNAP were amplified with primers overlapping into the intein. The three pieces, gel purified from a 1 % agarose gel, were then combined and amplified with primers at each end of T7 RNAP. This PCR product was digested, gel purified, and cloned. This fragment was then amplified to add the RBS and a 6His tag and cloned with the J23115 promoter. In order to create the second plasmid, the RFP reporter plasmid, we ligated the T7 promoter (annealed oligos) and RBS-RFP into the pSB3K3 plasmid. This plasmid, along with the plasmid containing the estrogen sensitive intein, were co-transformed into E. coli MACH cells and Top10 cells to be tested with estrogen.

Testing

The cells containing the sensor were grown overnight at 37°C. They were then diluted twenty fold in LB, and grown at 37 °C to OD 1.0 and treated with 17-β Estradiol from Acros Organics. The E.coli cells containing the sensor were tested with 0.5, 1, 2 and 10 ul of 10 mg/ml solution of estrogen in ethanol/glycerol/water (final 10-200 uM estrogen)incubated at 30 °C the optimum temperature for splicing. Samples of the culture were taken every 30 to 60 minutes for 4-6 hours and then sampled again the following morning. We also tested growth of the cells over time at different temperatures (20°C, 25°C, 30°C and 37°C). T7 RNAP without the intein was the positive control and splicing was rendered "Dead" by replacing the final residue of the C-intein, a asparagine, to a glutamine (N454Q). Cells expressing only YFP from the J23115 promter with and without the T7-RFP served as additional controls for background levels of RFP from cells and the T7-RFP plasmid.

Results

While we were able to successfully construct our sensor using overlap PCR, and a double transformation into E. coli MACH cells (a W strain that grows rapidly) and Top10 cells, we were unable to get a significant fluorescent signal from these cells in the presence of estrogen. Our model also predicted that we would not be able to detect any fluorescent signal. However, we were able to observe growth of the E.coli cells containing the sensor at 30 °C and 37 °C. In addition, we observed significantly higher fluorescence of the YFP and RFP in their respective controls.

The sensor did not function as expected however the T7 RNAP system worked exceptionally well. The RFP signal was over 400 times greater than when there was no T7 RNAP. This signal was greater at 37°C than 30°C and increased when the RBS-YFP was added at the 3' end of the T7 RNAP sequence.

A hypothesis for the increased YFP observed in the intein sensors compared to the T7 RNAP alone is the stalling of the ribosomes at the intein sequence giving increased access to the RBS of the YFP. This may result from the presence of eight, uncommonly used codons in the intein-ligand binding domain, which are AAG, AGA, AUA, CUA, CGA, CGG, CCC and UCG (Source: EMBL). The significant signal increase of RFP when the T7 RNAP has YFP added to the 3' end of the mRNA, may be due to mRNA stabilization by additional ribosome binding at the secondary RBS. These features make the system very attractive for producing a strong signal if we can achieve intein splicing in response to estrogen.

Data from Initial Testing

Source:

European Molecular Biology Laboratory (EMBL), The 8 least used codons in E. coli, yeast, Drosophila and primates. http://www.embl.de/pepcore/pepcore_services/cloning/choice_expression_systems/codons8/, October 16, 2014

In the following graphs,

• 115 YFP = Plasmid with J23115 promoter and YFP only
• YFP RFP = J23115-YFP plasmid and T7-RFP plasmid. There should be no T7 RNA polymerase.
• T7 RNA Polymerase = J23115-T7RNAP-YFP and T7-RFP plasmids. There is no intein.
• Dead Intein = J23115-T7RNAP with Dead Intein-YFP and T7-RFP plasmids. The dead intein should not be able to splice out to allow T7 RNA polymerase to form.
• Wild Type Intein = J23115-T7RNAP with Wild Type Estrogen Receptor Intein-YFP and T7-RFP plasmids. Ideally the intein would splice in the presence of estrogen.

Characterization of Sensor System

This graph shows the difference in red fluorescence from before the T7 RNAP was added (in the sample 115 YFP) and after it was added. We observed a significantly increased signal from RFP (over 400 times that of the RFP signal from the 115 RFP) after the T7 RNAP was added to the plasmid.

This graph shows the increased YFP signal in the sensor compared to T7 RNAP alone.

The sensor final sensor system contains a YFP coding region to report the amount of T7 RNA Polymerase that is produced. The following graphs show the fluorescence of the system and its controls before and after the YFP coding region was added in Top10 cells. Two graphs are shown because of the high magnitude of RFU in the RNAP sample after YFP was added. The following graphs show the amount of fluorescence in the red channel, so RFU is indicative of the amount of RFP that was synthesized in the two plasmid system.

Sensor Time Points

The fluorescence of the sensor and its controls were measure in the red and yellow channels with and without the presence of beta-estradiol over several time points.

*The magnitude of fluorescence of T7 RNA polymerase between the red and yellow channels was so large that they are presented on two different graphs.

Sensor in MACH Cells

Sensor in Top10 Cells

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