Difference between revisions of "Part:BBa K1033933"

Revision as of 01:40, 12 October 2022

asPink, pink chromoprotein

This chromoprotein from the coral Anemonia sulcata, asPink (also known as asCP or asFP595), naturally exhibits strong color when expressed. The protein has an absorption maximum at 572 nm giving it a pink/purple 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 asPink has significant sequence homologies with proteins in the GFP family.

Important: This part is not available in the registry yet, however, the same part is available from the registry with the standard RBS B0034: BBa_K1033927.

Usage and Biology

This part is useful as a reporter.

Contribution

Group: Linkoping_Sweden iGEM 2019
Author: Andreas Holmqvist, Leo Juhlin and Emmanuel Berlin
Summary: We studied the expression in of E.coli BL21(DE3) containing CBD-asPink (BBa_K3182100) protein with a constitutive promotor to see if the protein could be used for a pink/white screening. We also tested the bindning capacity of the CBD-asPink protein to a micro cellulose bandage and then studied the release mechanism.
Documentation: The expression system used contained the following parts:

• the BBa_B0034 Ribosome binding site
• the BBa_J23110 Constitutive promotor

Pink-white screening
To visualize the absorbance of AsPink on a petri dish, BL21 (DE3) colonies containing the pUC19-pCons-AsPink part (BBa_K3182100), which was used in the pink-white screening, were incubated for 16 hours in 37 °C. The pCons-AsPink construct was removed and (Magainin 2) and BBa_K3182104 (CHAP) was inserted into the vector. The pink colonies contain AsPink and indicate a religated pCons-AsPink. The white colonies indicate a successful ligation. Below (Figure 1) is a picture of the pink-white screening.

Figure 2. A: E. coli BL21 cells expressing the biobrick, incubated for 16 hours at 16°C at 80 rpm in 1L of LB-miller. B: Lysated (via sonication) BL21s which are expressing the biobrick. It is lysate from the culture above in figure1. Incubated for 16 hours at 16°C at 80 rpm in 1 litre of LB-miller. C:Centrifuged lysate of BL21 culture which express the biobrick. It is the same culture as figure 4 and 5. A pellet of non-lysated bacteria can be observed.

Figure 1. BL21 (DE3) bacteria used for pink-white screening. The white colonies indicate a successful ligation and the pink colonies is AsPink expressing bacteria which signals a false-positive ligation.

CBD-asPink bindning capacity

Figure 3. Lysate (via sonication) from E. coli BL21 was incubated with Epiprotect (microbial cellulose bandage) for 1h and washed thrice with 70% ethanol.

To test the bindning capacity of CBD-asPink (BBa_K3182000) the microbial cellulose bandage was suspended into sonicated lysate of E.coli BL21(DE3) with the expressed fusion protein (Figure 3) and incubated for 30 minutes. The bandage was then washed thrice in 70% ethanol which confirmed that the bindning of CDB-asPink was specific for the cellulose bandage.

Figure 4. To test the release mechanism of CBD-asPink, human thrombin and cleavage buffer was added to the bandage and incubated for 16 hours on an end to end rotator. The figure is the result after the incubation with a negative control with only cleavage buffer (left) and human thrombin and cleavage buffer (right).

CBD-asPink with thrombin cleavage
After the washes, human thrombin and cleavage buffer was added to the bandage with bound CBD-asPink to test the release mechanism of the fusion protein. The bandage was then incubated with the solution for 16 hours on an end to end rotator together with an negative control containing cellulose bandage and only cleavage buffer. After the incubation, the supernatant containing thrombin was pink while the negative control containing only cleavage buffer was transparent with a clear pink cellulose bandage (Figure 4). This indicates that the release mechanism work and the AsPink protein has successfully been released from the bandage into the supernatant.

asPink does not exist as CDS only but only as RBS-asPink (BBa_K1033927) and J23110-RBS-spisPink (BBa_K1033926).

iGEM2013 Uppsala: Expression of asPink in E. coli DH5alpha by promoter J23110 from medium copy plasmid pSB3K3 (left) or high copy plasmid pSB1K3 (right).

Source

The protein was first extracted and characterized by Lukyanov et. al. 2000 under the name asFP595 (GenBank: AAG02385.1). This version is codon optimized for E coli by Genscript.

References

[http://www.jbc.org/content/275/34/25879.short]Lukyanov, Konstantin A., et al. "Natural animal coloration can be determined by a nonfluorescent green fluorescent protein homolog." Journal of Biological Chemistry 275.34 (2000): 25879-25882.

[http://www.jbc.org/content/280/4/2401.short]Wilmann, Pascal G., et al. "Variations on the GFP Chromophore - A Polypeptide Fragmentation Within The Chromophore Revealed In The 2.1-Å Crystal Structure Of A Nonfluorescent Chromoprotein From Anemonia Sulcata." Journal of Biological Chemistry 280.4 (2005): 2401-2404.

Contribution

Group: Worldshaper-Nanjing 2022iGEM
Author: Ziqi Huang，Yunzheng Tian
Summary: Characterization of the expression of aspink in E.coli strain BL21 transformed with pET-29a-aspink plasmid, the purification of aspink protein and the bioinformatic information of aspink protein.

Characterization from Worldshaper-Nanjing

We obtained the plasmid vector containing this part, pET-29a-aspink plasmid, from iGEM team iGEM13_Uppsala. We transformed this plasmid into E.coli strain BL21, and induced aspink expression. We extracted and purified the aspink protein by sonication, ultrafiltration, and Ni-affinity chromatography. With bioinformation analysis using Swiss Model, we predicted the structure of aspink protein and analyzed detailed characteristics of the protein. The relative molecular mass was measured by mass spectrometry.

Experiments Results

1.Induced expression of aspink in E. coli BL21 transformed with pET-29a aspink plasmid

The growth performance of the BL21 bacteria samples transformed with pET-29a-aspink plasmid was measured. Two samples with OD value around 0.6 were used as the control group and the induced group, respectively. IPTG was added to induce aspink expression (Figure 1). Protein was extracted and purified from the two groups by sonication, ultrafiltration, and Ni-affinity chromatography (Figure 2).

Figure 1. Comparison of induced group (left) and control group (right) for the induced expression of pET-29a-aspink plasmid in BL21

Figure2. Purification of aspink protein with nickel column

2.SDS-PAGE verification of aspink expression

SDS-PAGE experiment verified the successful expression of aspink protein induced in BL21 (Figure 3). The size of aspink is predicted around 26 kDa.

3.Aspink Bioinformation Analysis

With bioinformation analysis using Swiss Model, we predicted the structure of aspink protein (Figure 4). Further the detailed characteristics of the protein were analyzed as shown in Figure 5.

4.Mass spectrometry of the relative molecular mass of aspink

We used mass spectrometry to measure the relative molecular mass of aspink protein (Figure 6). The relative molecular mass measured was roughly 25.9 kDa, consistent with our prediction. The majority 19 kDa implicates that the expressed protein was not intact.

Sequence and Features