Difference between revisions of "Part:BBa K1033923"

Latest revision as of 20:19, 19 October 2021

spisPink, pink chromoprotein (incl RBS, J23110)

This chromoprotein from the coral Stylophora pistillata, spisPink (also known as spisCP), naturally exhibits strong color when expressed. The protein has an absorption maximum at 560 nm giving it a pink color visible to the naked eye. The strong color is readily observed in both LB or on agar plates after less than 24 hours of incubation. The protein spisPink has significant sequence homologies with proteins in the GFP family.

Usage and Biology

This part is useful as a reporter.

iGEM2013 Uppsala: The images above show E coli constitutively expressing spisPink BBa_K1033925 from the high copy plasmid pSB1C3 from the promoters J23110 (left) and J23106 (right).

Characterisation

Team: Humboldt_Berlin 2019

ExPASy ProtParam Results:

Number of amino acids: 223

Molecular weight: 24815.62 Da

Theoretical pI: 8.23

Total number of negatively charged residues (Asp + Glu): 26

Total number of positively charged residues (Arg + Lys): 28

Extinction coefficients:

Ext. coefficient 24660 M-1 cm-1

Abs 0.1% (=1 g/l) 0.994, assuming all pairs of Cys residues form cystines

Ext. coefficient 24410 M-1 cm-1

Abs 0.1% (=1 g/l) 0.984, assuming all Cys residues are reduced

Instability index: The instability index (II) is computed to be 26.85. This classifies the protein as stable.

Aliphatic index: 67.35

Grand average of hydropathicity (GRAVY): -0.352

In order to measure the absorbance spectrum of spisPink we transformed the construct (consisting of BBa_J23110 Promotor, RBS and spisPink coding sequence) into E. coli (DH10B Competent Cells). After cultivation and we lysed the harvested cells according to this protocol.

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  Liquid culture of E. coli expressing spisPink

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  Cell pellet after centrifugation

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  Cleared cell lysate

The absorbance spectrum was measured for 24 samples of 150 µl lysate in 96-well plate on TECAN Plate Reader Infinite 200 Pro. In figure 1 you can see the absorbance spectrum including the respective standard deviation with a peak at 562 nm (compared to an excitation maximum of 560 nm in the literature [2]).

Figure 1. spisPink absorbance spectrum

Contribution

Group: Linkoping_Sweden iGEM 2021
Author: Ewelina Bladh
Summary: In this contribution, we recorded color development in colonies of E.coli cells of strain XL1. In addition to studying the color development, we confirmed spisPink's absorbance maximum wavelength to show reproducibility.

Color Development
In order to determine the progression of color development in colonies, E.coli cells (XL1) were plated onto LB-Miller agar plates containing a concentration of 25 ug/mL chloramphenicol. The cells were plated in two ways after transformation. The first by plating 100 µL immediately from the liquid culture. The second by first centrifuging the culture, discarding 90% of the supernatant and resuspending the cell pellet in the remaining 10%, followed by plating 100 µL of the resuspension. This results in what is from now on referred to as the diluted and concentrated plate, respectively.

These plates were incubated in 37°C for the following four days, where they were checked upon each day. Figure 1, displayed below, shows the diluted and concentrated plates during this four day study. The illustration of the diluted plate shows that after 12 hours, no color can be seen to the naked eye. This changes over the course of the next 24 hours, where at 36 hours the colonies are clearly visible. The color intensity increases over the next two days. The same can be said about the concentrated plate, but unlike the diluted plate the concentrated plate shows weak indications of colored colonies already at 12 hours.

Absorbance and Fluorescence
Single colonies displaying a pink color were picked from the diluted plates to create liquid cultures. The liquid cultures consist of 5 mL LB-Miller media and a concentration of 25 µg/mL chloramphenicol. The liquid cultures were incubated at 37°C and 200 rpm for the next two days to ensure protein expression. After incubation, the liquid cultures were centrifuged until a pellet formed. The supernatant was discarded, and the remaining pellet was placed in a -20°C freezer. Cell lysis was done using both water and sonication. The frozen pellet was thawed and 5 mL autoclaved dH₂O was added, followed by vortex treatment to resuspend the pellet. Sonication on ice was the next step taken to lyse any cells that did not lyse upon the addition of water. The amplitude of the sonicator was set to 30%, 30 seconds pulsing followed by 30 seconds rest for a total of 2 minutes. Following sonication, the cultures were centrifuged until a pellet formed whereupon the supernatant was transferred to a clean 15 mL Falcon tube and the pellet was discarded. The supernatant of spisPink can be see to the left in Figure 2. Simultaneous experiments with other chromoproteins was also made, their supernatant can be seen with spisPink to the right in Figure 2. The following chromoproteins were also assessed in the same way as spisPink: tsPurple (BBa_K1033903), amajLime (BBa_K1033914), meffBlue (BBa_K1033900), gfasPurple (BBa_K1033917), and asPink (BBa_K1033926).

As a control, plates were also made with untransformed XL1-cells. A colony was picked from the diluted plate to start a liquid culture, followed by lysis like the cells transformed with spisPink. Treatment of control and experimental cells was identical apart from the control culture not being exposed to antibiotics. The supernatant of the control can be seen to the right in Figure 2 along with other chromoproteins.

In order to measure the absorbance, 100 µL supernatant of spisPink and 100 µL supernatant of the control were pipetted into a 96-well plate and measurements were made with BMG Clariostar plate reader. Measurements of absorbance were taken every 2 nm in the range 300 nm to 850 nm. This interval was chosen to include the entire visual spectra including margins. The data yielded from the measurements were subsequently baselined by subtracting the control's absorbance values from spisPink's absorbance values followed by normalization. The resulting graph for spisPink's absorbance can be seen in Figure 3 below. The absorbance maximum of spisPink proved to occur at approximately 562-564 nm. This result is the same as the team of Humboldt_Berlin of 2019 achieved (see above).

When examining the supernatant extracted from spisPink-expressing cells on a UV-table, no fluorescence was detected. The chromoprotein amajLime (BBa_K1033914) was studied at the same time, functioning as a positive control, and the lysate from the control functioned as a negative control.

Source

Stylophora pistillata. The protein was first extracted and characterized by Alieva et. al. under the name spisCP (GenBank: ABB17971.1). This version is codon optimized for E coli by Genscript.

References

[http://www.ncbi.nlm.nih.gov/pubmed/18648549] Alieva, Naila O., et al. "Diversity and evolution of coral fluorescent proteins." PLoS One 3.7 (2008): e2680.

[1] Liljeruhm, Josefine et al. “Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology.” Journal of biological engineering vol. 12:8. 10 May. 2018, doi:10.1186/s13036-018-0100-0

Sequence and Features