Difference between revisions of "Part:BBa K1979004"
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| + | <p class="title" style="font-size:20px;">Construct</p><br/> | ||
| + | <p>The GFP sequence is cloned from eGFP plasmid through PCR, using the primers designed and synthesized based on the sequence of PBP5, with restriction enzyme sites XbaI, promoter BBa_J23101 and ribs added on the upstream primer, and BamH I added on the downstream primer. The PCR sequence and our PBP5 generator (BBa_K1979003) are digested by XbaI and BamHI, after which they are purified and ligated, and become our new construct.</p> | ||
<img src="https://static.igem.org/mediawiki/parts/3/30/T--SDSZ_China--gfp-pbp_PCR.jpg" style="width:60%;"/> | <img src="https://static.igem.org/mediawiki/parts/3/30/T--SDSZ_China--gfp-pbp_PCR.jpg" style="width:60%;"/> | ||
<p class="text">Figure1. PCR check for BBa_K1979004. GFP sequence is magnified by primers, with a size of 716 bps (red box).</p> | <p class="text">Figure1. PCR check for BBa_K1979004. GFP sequence is magnified by primers, with a size of 716 bps (red box).</p> | ||
Revision as of 03:38, 30 October 2016
GFP-PBP5
This device codes for the GFP-PBP5 fusion protein.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 135 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1892
Illegal BamHI site found at 168
Illegal XhoI site found at 2100 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1206
Illegal AgeI site found at 1808 - 1000COMPATIBLE WITH RFC[1000]
Construct
The GFP sequence is cloned from eGFP plasmid through PCR, using the primers designed and synthesized based on the sequence of PBP5, with restriction enzyme sites XbaI, promoter BBa_J23101 and ribs added on the upstream primer, and BamH I added on the downstream primer. The PCR sequence and our PBP5 generator (BBa_K1979003) are digested by XbaI and BamHI, after which they are purified and ligated, and become our new construct.
Figure1. PCR check for BBa_K1979004. GFP sequence is magnified by primers, with a size of 716 bps (red box).
Figure2. M: Marker; 1: the initial E.coli suspension; 2: deposit of E.coli after centrifugation; 3: deposit of disrupted E.coli; 4: supernatant of disrupted E.coli; 5: W1; 6: W2; 7: W3; 8-11: Elute.
The PBP-GFP was purified through Ni-chelating affinity chromatography. The target protein, PBP-GFP, is marked in the red box, has a molecular weight of 60kDa. As demonstrated in channel 1-4 that protein is identified at the position of 60kDa, PBP-GFP is expressed in the E.coli; as demonstrated in channel 8-11 that the target protein is found in all 4 elutes, we have successfully purified PBP-GFP from the E.coli.
Figure3. M: Marker 1-13: protein through Q Sepharose Fast Flow.
The target protein from channel 8-11 in figure 2 was purified through Q Sepharose Fast Flow. Proteins in channel 3-5 have clear marks at 60kDa, indicating our construction can express the GFP-PBP we desire.
Proof of Function Through Experiment:
We incubate penicillin on the coated wells on the plate, add free penicillin (with varied concentration listed in Table 1) and PBP5-GFP, and allow them to react, during the process of which bound penicillin and free penicillin in the sample compete for PBP5-GFP, leading to the result that certain amount of PBP5-GFP would bind to free penicillin instead of the bounded penicillin. Testing the intensity of green fluorescence in the liquid sample, we are able to identify the amount of PBP-GFP, which also indicates the amount of free penicillin in the sample.
We conduct three different competitive binding experiments: the first two experiments enable us to determine one proper condition under which PBP works the best. And we use preprocessed milk sample in the third experiment, proving that our method can work under real life condition. The details and data are provided below.
Results:
The first competitive binding experiment:
Interpretation:
In the first competitive binding experiment, we compare the result of applying different concentrations of penicillin. By calculating the correlation of each experiment, we discovered that 25 times diluted PBP-GFP5) leads to data with highest relativity. (See more in protocol)
The second competitive binding experiment:
Interpretation:
The second experiment is conducted under the condition of 25 times diluted PBP5-GFP solution. By analyzing the data and applying the Pearson correlation coefficient, we are able to determine the excitation wavelength that ensure the highest relativity is be 405nm. (See more in protocol)
The preprocessing of milk:
We add milk sample into the centrifuge tube and add the equal amount of tris-saturated phenol into the milk. Then we add four times volume of chloroform into the mixture, and centrifuge the tube at 10000rpm for 5 min. (See Protocol) The result of the preprocessing was showed in the picture above.
Demonstration:
Interpretation:
The last competitive binding experiment is conducted under the condition of 25 times diluted PBP5-GFP solution and preprocessed milk (containing PBP-GFP) and 405nm excitation wavelength (See more in protocol). The results indicate that the fluorescence intensity measured and penicillin concentration has a correlation factor of 0.8212211, which means the two factors is strong correlation, proving that our method doesn’t just work under the most simplified conditions and has the ability to detect penicillin in milk samples.