Difference between revisions of "Part:BBa K1692032"

Latest revision as of 03:16, 22 October 2019

amilCP blue chromoprotein with RBS and promoter

This plasmid contains the amilCP blue chromoprotein gene found in part BBa_K592009 and the RBS and promoter from part BBa_K608002. For more information about this gene, please refer to the BBa_K592009 part page.

Usage and Biology

The chromoprotein amilCP is part of a family of GFP-like fluorescent proteins derived from reef-building corals of the class Anthozoa (Alieva et al., 2008). Similar to the GFP family of proteins, the β-barrel of amilCP encloses a chromophore (Tafoya-Ramírez et al., 2018). Along with other coral chromoproteins, amilCP is non-fluorescent and forms a tetramer, resulting in a fairly stable protein structure (Alieva et al., 2008; Tafoya-Ramírez et al., 2018). These GFP-like fluorescent proteins give corals their vivid and varied colors (Alieva et al., 2008).

Isolated from the species Acropora millepora, amilCP was first described in 2008, and is characterized by a strong color due to its very high molar extinction coefficient of 87 600 (Alieva et al., 2008). Its maximum excitation wavelength is 588nm (Alieva et al., 2008). Interestingly, amilCP’s max absorption is shifted into the red spectrum by ~10nm (592nm), hence appearing more blue than purple to the naked eye (Alieva et al., 2008). Such blue color is due to 2 mutations resulting in amino acid substitutions (Alieva et al., 2008).

Experiments

Protein Purification and SDS-PAGE gel electrophoresis

Purified samples of protein allow further experiments to characterize their biological functions and interactions. Because this protein is not tagged with a short peptide sequence for affinity chromatography, the easiest method for purification of this protein may be by size-exclusion chromatography. Here, we propose a method for purification of amilCP via size-exclusion chromatography.

The following protocol uses a 25mL culture of amilCP transformed into DH5α E. coli cells. On this SDS-PAGE gel electrophoresis, the protein is visualized to be between 22kDa and 25kDa which appears to be slightly smaller than the theoretical mass of 25.4kDa (Tafoya-Ramírez et al., 2018). However, we believe it nonetheless represents the purified protein as the ladder runs slightly diagonally, the collected fraction was pigmented and the SDS-PAGE shows a purified protein.

Figure 1: amilCP purified from E. coli following the adjacent protocol was loaded onto SDS-PAGE gel electrophoresis. The ladder on the left (NEB Blue Protein Standard Broad Range) spans from 11kDa to 190kDa in the following increments from bottom to top: 11kDa, 17kDa, 22kDa, 25kDa, 32kDa, 46kDa, 58kDa, 75kDa, 100kDa, 135kDa, 190kDa.

Buffer Preparation

1M phosphate buffer (40ml):

• 2.596g KH₂PO₄
• 3.644g K₂HPO₄
• Top off with distilled water to 40ml

50mM (0.05M) Phosphate Buffer (40ml):

• 100mM NaCl
• 25mM sucrose
• 1mM hexadecyltrimethylammonium bromide
• 2ml of 1M phosphate buffer previously made

Cell Lysis Solution (40ml, experimental):

• 2ml of 1M phosphate buffer
• Top off with distilled water to 40ml

Cell Lysis and Protein Extraction (keep samples on ice):

1. Remove supernatant and resuspend in 1ml dH₂O
2. Transfer to 1.5ml centrifuge tubes
3. Centrifuge at 14500rpm for 15min
4. Remove supernatant and resuspend in cell lysis solution
5. Vortex to lyse cells and release protein into solution
6. Centrifuge at 13000rpm for 5 min
7. Supernatant should be colored (if chromoprotein is present). Transfer this supernatant to new 1.5ml centrifuge tube to separate protein from cell debris.Likewise, if the culture broth appears to be sufficiently colored due to the presence of protein in solution, this can be used instead of lysed cells.

Gel Preparation

1. Weigh an appropriate amount of separation gel. For Sephadex G-100 Superfine, every gram of dry mass produces between 15ml to 20ml of gel. Since Sephadex gels are very “spongy” prepare a larger volume than needed as it will compress in the column.
2. Dissolve the dry gel in an appropriate amount water to the gel weighed.
3. Degas the gel by heating the solution to 90°C for 5 hours.
4. Once the gel is degassed and ready, it should be slightly viscous.

Column Packing

1. Pour the column. Care should be taken to pour the entire column in one session as adding more gel afterwards will disrupt the matrix. **Pour more gel than is necessary into the column as the mobile phase added afterwards will compress the gel be by a significant amount**.
2. Pack the column by washing it with a volume of wash buffer equal to twice the volume of the column being used.
3. Leave approximately 1mm of wash buffer above the gel bed and store in refrigerator (4^oC)when not in use.

Purification

1. Set 500μl (may vary depending on size of column) of sample on the column and let run until the aliquot has completely entered the gel.
2. When necessary add a small amount of wash buffer to the column to continue elution. Avoid adding large quantities of wash buffer as it will further compress the Sephadex Gel.
3. Collect aliquots in centrifuge tube. These samples can be further analysed.

Troubleshooting if Column Stops Running

1. The column is over packed. Remove the gel from the column and combine it with the remaining unused gel previously prepared. This homogenized the gel and reproduces a uniform matrix.
2. The column is poisoned. When working with excessively contaminated samples, they may bind permanently to the gel and prevent further elution. To fix, remove the upper portion of the gel and dispose of it. Combine the remaining gel to the stock and repack the column.
3. If the sample is highly contaminated, perform a run on a smaller column to clean out most of the impurities and then run it on the true column.

Circular Dichroism of amilCP

Figure 2: A circular dichroism spectrum of amilCP indicates that the secondary structure is predominantly composed of β-sheets.

Purpose

The interaction of circularly polarized light with chiral molecules causes light of different rotation (clockwise or counter clockwise) to be absorbed unevenly (Van Holde et al., 2006). Such effect occurs in the far UV range of light due to the secondary structures within a protein. Different types of secondary structures produce different spectra that is unique to the structure. Of the two major secondary structures, α-helices produce a double minimum at 222nm and 208nm (Van Holde et al., 2006). This is followed by a sharp increase in ellipticity towards 190nm. Β-sheets result in only a single minimum around 215nm and a maximum around 198nm, although to a lesser extent than α-helices (Van Holde et al., 2006). The circular dichroism spectrum of AmilCP was analyzed to determine the major secondary structure features of AmilCP. This can be used to determine the purity of AmilCP in future purified samples of AmilCP along with providing insight on the specific interactions that contribute to the protein’s stability as denaturation results in the loss of secondary structure which will be reflected as a loss of ellipticity of circularly polarized light passing through the sample.

Methods

amilCP (BBa_K1692032) transformed into DH5-α cells was grown in an LB liquid culture for 3 days. The resulting culture was centrifuged and resuspended in dH₂O. The sample was pelleted again and resuspended in lysis buffer. After centrifugation, the supernatant was purified by size-exclusion chromatography using a Sephadex G-100 Superfine column. The sample was purified in two runs, the first to separate the larger molecules and lysis buffer from the protein and a second run to further purify the protein. As a chromoprotein, fractions were preserved based on their blue color, indicating the presence of amilCP. The fractions were then analyzed by circular dichroism the determine optical rotation of circularly polarized light as it interacts with the secondary structures within the protein.

Results

The circular dichroism spectrum of amilCP indicates that the secondary structure is predominantly composed of β-sheets as indicated by the single minimum around 217nm and the maximum above 195nm. The minimal broadening towards 210nm and the second maximum at 190nm suggests that there are discrete α-helix structures in the proteins but are not a significant feature of this protein. The impact of the β-sheets on the ellipticity of circularly polarized light prevents any smaller features from being characterized. Furthermore, a negative of polarized light followed by a positive rotation of polarized light suggests that the β-sheets are arranged in an anti-parallel fashion throughout the protein.

amilCP Growth Curve in E. coli

Estimating the time necessary for amilCP to start expressing and reach maximum expression rates in E. coli cells can be extremely useful for optimization of protocols or experiments that utilize this chromoprotein and depend on a strong color signal. To do so, continuous monitoring of absorbance at specific wavelengths (i.e. amilCP's maximum absorption wavelength at 592nm (Alieva et al., 2008)) can help determine changes in protein concentration over time within cell cultures, and give an estimate at which time points amilCP expression is highest in E. coli.

Figure 3: replicates of cells expressing amilCP (first row) are visibly colored. The control cells are located on the second row and the LB blank on the third.

Methods

Here, we cultured amilCP transformed in DH5α cells in LB medium for 5 hours (before any visible color change appeared) and concentrated the cells to an OD₆₀₀ of 0.9. Then, 180uL of culture was transferred onto a 96-well plate (3 replicates) to be monitored over the span of 48 hours inside a plate reader at 37^oC with shaking. Shaking is important as lack thereof will result in protein accumulation at the bottom of the well and skew absorbance reading. It is also important to seal the plate to prevent evaporation. To blank the cultures, replicates of LB medium was used, whereas the negative controls were composed of DH5α cells in LB medium. The data points were recorded and plotted. Absorbance was measured at 595nm due to restrictions in equipment, but to account for this, the absorbance of control cells was subtracted.

Results

The data points used to plot the expression curve were collected up until the ~10th hour where a clear plateau is reached. The grey zones surrounding the curve represents the standard deviation between the 3 replicates. An important difference can be noticed between absorbance of amilCP-expressing cells and control cells at 595nm (figure 4). Net increase in absorbance at 595nm due to expression increase of amilCP is shown in figure 5 where absorbance of control cells was subtracted. Figure 5 shows a peak in absorbance before the 5 hour culture mark in the 96-well plate. Therefore, in total, our transformed cells required less than 10 hours (5 hours pre-measurement plus 5 hours during measurement) to reach the maximum level of amilCP expression.

Figure 4: comparison of absorbance at 595nm of cells expressing the chromoprotein amilCP and untransformed DH5α cells.

Figure 5: net increase in absorbance at 595nm in cells expressing amilCP (control subtracted), suggesting the the rate of expression of amilCP peaks before the 10 hour mark.

References

1. Alieva, N. O., Konzen, K. A., Field, S. F., Meleshkevitch, E. A., Hunt, M. E., Beltran-Ramirez, V., … Matz, M. V. (2008). Diversity and evolution of coral fluorescent proteins. PLoS ONE, 3(7). https://doi.org/10.1371/journal.pone.0002680
2. Tafoya-Ramírez, M. D., Padilla-Vaca, F., Ramírez-Saldaña, A. P., Mora-Garduño, J. D., Rangel-Serrano, Á., Vargas-Maya, N. I., … Franco, B. (2018). Replacing Standard Reporters from Molecular Cloning Plasmids with Chromoproteins for Positive Clone Selection. Molecules (Basel, Switzerland), 23(6). https://doi.org/10.3390/molecules23061328
3. Van Holde, K.E; et. al. Principles of Physical Biochemisty. 2nd ed. Pearson. Upper Saddle River, NJ. 2006

Sequence and Features