Difference between revisions of "Part:BBa K1979003"
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===Usage and Biology==-->
 
===Usage and Biology==-->
 
<html>
 
<html>
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<img src="https://static.igem.org/mediawiki/parts/c/c9/T--SDSZ_China--PBP1_.jpg" style="width:60%;"/>
 
<img src="https://static.igem.org/mediawiki/parts/c/c9/T--SDSZ_China--PBP1_.jpg" style="width:60%;"/>
 
<p class="text">Figure1. Double enzyme digestion check for BBa_K1979003. PBP5 has an expected size of 1200 bps (red box). </p>
 
<p class="text">Figure1. Double enzyme digestion check for BBa_K1979003. PBP5 has an expected size of 1200 bps (red box). </p>
 +
 +
<br/><p>PBP5 causes the hydrolysis of tripeptide Lys-Ala-Ala(KAA) when active and not bound with penicillin, releasing one alanine and exposing one amino group.[3] We measure its hydrolysis by the amino group, which changes the color of KAISER, a substance that reacts with free amino acids to yield CO2, NH3, and an aldehyde, the NH3 further yielding a colored product (diketohydrindylidene-diketohydrindamine, a bi-indanedione derivative also called Ruhemann purple), yielding the color purple. With more penicillin present, more PBP5 becomes inactive, and the purple gets lighter.
 +
                </p><br/>
 +
                    <p class="title">Description:</p><br/>
 +
                    <p>Many poplypetides with a d-ala-d-ala end can be the substrate for PBP5, diacetyl-L-LysD-Ala-D-Ala (Ac2-L-Lys-D-Ala-D-Ala, Ac2-KAA) is the common substrate used to detect PBP5’s activity[2]. The PBP5 causes  Ac2-KAA to hydrolyze, releasing one alanine and exposing one amino group. If bound with penicillin, PBP5 loses activity. And the protein is very sensitive to the penicillin in the liquid, so this mechanism is applicable as we try to determine the amount of penicillin in a low concentration system. the amino group, which turns Kaiser blue.
 +
                    </p><br/>
 +
                    <p>We mix 5 times diluted supernatant of the bacteria (treated with French pressure cell press and centrifugation at 12000rpm for 5min), with 100 times diluted Ac2-KAA. The mixture is equally devided into 8 centrifuge tubes, and different concentrations of penicillin (indicated below) are added. By applying the KAISER test, we were able to detect the amount of penicillin in each sample. (<a href="https://static.igem.org/mediawiki/2016/4/41/T--SDSZ_China--protocol4.pdf">See protocol</a>)
 +
                    </p><br/>
 +
                    <p class="title">Results of KAISER Test</p>
 +
                    <br/>
 +
                    <img src="https://static.igem.org/mediawiki/2016/d/de/T--SDSZ_China--kaiser.png" style="height: auto; width: 30vw; left:0;">
 +
                    <br/><br/><br/>
 +
                    <img src="https://static.igem.org/mediawiki/2016/5/59/T--SDSZ_China--ptable6.png" style="height: auto; width: 50vw; left:0;">
 +
                    <br/><br/><br/>
 +
                    <img src="https://static.igem.org/mediawiki/2016/2/2c/T--SDSZ_China--pfigure5.png" style="height: auto; width: 50vw; left:0;">
 +
                    <br/><br/><br/>
 +
                    <p class="title">Interpretation:</p>
 +
                    <br/>
 +
                    <p>According to the graph above, the horizontal axis represents the number of the sample and the vertical axis shows the optical density of each sample. By applying Pearson correlation coefficient, the result is 0.931032451, which means the two factors have strong correlation. As the concentration of penicillin increased, the value of optical density decreased, which proves the validity of our approach.
 +
                    </p><br/>
 +
                    <p class="title">Optimization:</p>
 +
                    <br/>
 +
                    <p>In order to apply the Ac2-KAA and KAISER test to milk samples, we need to reduce the interference of the amino groups on milk proteins. We designed a part that generates PBP5-CBD (Cellulose Binding Protein) to bind PBP5 onto the nano-cellulose fiber aerogel.[5] We plan to use the aerogel to collect the proteins added in the sample, and conduct the KAISER test with the rinsed aerogel and Ac2-KAA solution.
 +
                    </p>
 +
                        <br/><p class="title">Part proof of function:</p>
 +
                    <br/>
 +
                    <img src="https://static.igem.org/mediawiki/2016/f/f8/T--SDSZ_China--cbd.png" style="height: auto; width: 20vw; left: 0;">
 +
                    <br/><br/><br/>
 +
                    <p>The CBD-PBP5 protein(red box) is expressed, with a molecular weight of 53kDa. E.coli in channel 3 is a blank comparison.
 +
                    </p><br/>
 +
                    <p class="title">Preparation of nano-cellulose fiber aerogel:</p>
 +
                    <br/>
 +
                    <p>Nano-cellulose fiber aerogel’s porous structure ensures great absorptivity, which makes it an ideal carrier for our CBD-PBP5 protein. We prepare nano-cellulose fiber aerogel using conifer wood pulp board, through TEMPO(2,2,6,6-tetramethyl-1-piperidinyloxy) oxidation, high-pressure homogenization, and freeze drying. (<a href="https://static.igem.org/mediawiki/2016/5/54/T--SDSZ_China--nano_cellulose_fiber_.pdf">See protocol</a>)
 +
                    </p><br/>
 +
                    <img src="https://static.igem.org/mediawiki/2016/9/90/T--SDSZ_China--aerogel_.jpg" style="height: auto; width: 50vw; left: 0;">
 +
                    <br/><br/><br/>
 +
                    <p class="title">Future Plans:
 +
                    </p><br/>
 +
                    <p>With the nano-cellulose fiber aerogel prepared and the CBD-PBP5 protein harvested, we plan to take advantage of the aerogel’s excellent absorption and use it to collect CBD-PBP5 proteins after they are added to the sample. Then we plan to add the aerogel into the Ac2-KAA solution, so that the remaining active PBP5 on the aerogel can cause Ac2-KAA’s hydrolysis, and KAISER test can be done with the solution after the reaction. We anticipate that without the interference of amino groups on milk proteins, it is possible that the KAISER test can be performed on milk samples and be utilized in real life.
 +
                    </p>
 +
 
</html>
 
</html>
  
 
<partinfo>BBa_K1979003 parameters</partinfo>
 
<partinfo>BBa_K1979003 parameters</partinfo>

Revision as of 01:12, 30 October 2016

PBP5 coding device (promoter+PBP5+T7 terminator)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 135
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1178
    Illegal BamHI site found at 168
    Illegal XhoI site found at 1386
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 492
    Illegal AgeI site found at 1094
  • 1000
    COMPATIBLE WITH RFC[1000]

Figure1. Double enzyme digestion check for BBa_K1979003. PBP5 has an expected size of 1200 bps (red box).


PBP5 causes the hydrolysis of tripeptide Lys-Ala-Ala(KAA) when active and not bound with penicillin, releasing one alanine and exposing one amino group.[3] We measure its hydrolysis by the amino group, which changes the color of KAISER, a substance that reacts with free amino acids to yield CO2, NH3, and an aldehyde, the NH3 further yielding a colored product (diketohydrindylidene-diketohydrindamine, a bi-indanedione derivative also called Ruhemann purple), yielding the color purple. With more penicillin present, more PBP5 becomes inactive, and the purple gets lighter.


Description:


Many poplypetides with a d-ala-d-ala end can be the substrate for PBP5, diacetyl-L-LysD-Ala-D-Ala (Ac2-L-Lys-D-Ala-D-Ala, Ac2-KAA) is the common substrate used to detect PBP5’s activity[2]. The PBP5 causes Ac2-KAA to hydrolyze, releasing one alanine and exposing one amino group. If bound with penicillin, PBP5 loses activity. And the protein is very sensitive to the penicillin in the liquid, so this mechanism is applicable as we try to determine the amount of penicillin in a low concentration system. the amino group, which turns Kaiser blue.


We mix 5 times diluted supernatant of the bacteria (treated with French pressure cell press and centrifugation at 12000rpm for 5min), with 100 times diluted Ac2-KAA. The mixture is equally devided into 8 centrifuge tubes, and different concentrations of penicillin (indicated below) are added. By applying the KAISER test, we were able to detect the amount of penicillin in each sample. (See protocol)


Results of KAISER Test











Interpretation:


According to the graph above, the horizontal axis represents the number of the sample and the vertical axis shows the optical density of each sample. By applying Pearson correlation coefficient, the result is 0.931032451, which means the two factors have strong correlation. As the concentration of penicillin increased, the value of optical density decreased, which proves the validity of our approach.


Optimization:


In order to apply the Ac2-KAA and KAISER test to milk samples, we need to reduce the interference of the amino groups on milk proteins. We designed a part that generates PBP5-CBD (Cellulose Binding Protein) to bind PBP5 onto the nano-cellulose fiber aerogel.[5] We plan to use the aerogel to collect the proteins added in the sample, and conduct the KAISER test with the rinsed aerogel and Ac2-KAA solution.


Part proof of function:





The CBD-PBP5 protein(red box) is expressed, with a molecular weight of 53kDa. E.coli in channel 3 is a blank comparison.


Preparation of nano-cellulose fiber aerogel:


Nano-cellulose fiber aerogel’s porous structure ensures great absorptivity, which makes it an ideal carrier for our CBD-PBP5 protein. We prepare nano-cellulose fiber aerogel using conifer wood pulp board, through TEMPO(2,2,6,6-tetramethyl-1-piperidinyloxy) oxidation, high-pressure homogenization, and freeze drying. (See protocol)





Future Plans:


With the nano-cellulose fiber aerogel prepared and the CBD-PBP5 protein harvested, we plan to take advantage of the aerogel’s excellent absorption and use it to collect CBD-PBP5 proteins after they are added to the sample. Then we plan to add the aerogel into the Ac2-KAA solution, so that the remaining active PBP5 on the aerogel can cause Ac2-KAA’s hydrolysis, and KAISER test can be done with the solution after the reaction. We anticipate that without the interference of amino groups on milk proteins, it is possible that the KAISER test can be performed on milk samples and be utilized in real life.