Difference between revisions of "Team:Fudan/Part Collection"

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   <h1>Our part collection offers a complete array of parts for further researchers to develop other probiotic secreted proteins/peptides.</h1>
 
   <h1>Our part collection offers a complete array of parts for further researchers to develop other probiotic secreted proteins/peptides.</h1>
 
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     <p>In the collection, we have secreted mcbABCD to help its colonization in the gut, and secreted CaAP to facilitate calcium uptake by gut cells. We test all parts used in these two circles: 5 signal peptide for secretion, 14 promoters for different strength expression (previously publish and available in addgene.org but not in the Registry), IPTG inducible promoter driven various protein coding sequences (confirmed individually before assembled together), and quorum-sensing components. </p>
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     <p>In the collection, we have secreted mcbABCD to help its colonization in the gut, and secreted CaAP to facilitate calcium uptake by gut cells. We test all parts used in these two circles: 5 signal peptide for secretion, 14 promoters for different strength expression (previously publish and available in <a href="https://addgene.org" target="_blank">addgene.org</a> but not in <a href="http://parts.igem.org/Main_Page" target="_blank">the Registry</a>), IPTG inducible promoter driven various protein coding sequences (confirmed individually before assembled together), and quorum-sensing components. A file contains those parts could be
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     <a href="https://static.igem.org/mediawiki/2020/d/d5/T--Fudan--part_collection_list.xlsx "> Click here to see more details</a>
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     <a href="https://static.igem.org/mediawiki/2020/d/d5/T--Fudan--part_collection_list.xlsx ">downloaded (updated on 2020-10-27)</a>.</p>
 
     <p>We not only provide sequences and references for the parts, but also experimental results and procedures. We compete for both the Gold Medal criterion #7 and the Best Part Collection prize with this page. </p>
 
     <p>We not only provide sequences and references for the parts, but also experimental results and procedures. We compete for both the Gold Medal criterion #7 and the Best Part Collection prize with this page. </p>
 
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  <h2>Five signal peptide for secretion</h2>
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    <p>We have constructed plasmids using CaAP or MBP (Maltose Binding Protein, a protein that is expressed in the cytoplasm of <i>E.coli</i>) with five signal peptides (CaAP fused with signal peptides: BBa_K3606031~BBa_K3606035; MBP fused: BBa_K3606037~BBa_K3606041). We have quickly test several of them: the total production and relative amount being secreted. We show that OmpA, one of our signal peptides, could lead MBP to secret. Our results consist with the literature that for a specfic protein all possible signal peptides should be test for best results.</p>
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  </div>
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    <img src="https://static.igem.org/mediawiki/2020/e/e9/T--Fudan--2020-10-27-gel3.jpg" alt="1027 gel3" />
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    <h6>Figure 1. TCA precipated culture media (lane 8/with 9/no IPTG OmpA-MBP)</h6>
 +
    <p>Lane 8 and 9, bacteria DH5a with OmpA-MBP, culture to OD 0.6 and induced with 1mM IPTG for 6 hours. Then, the culture media was collected and precipated with TCA. Samples were run on 17% SDS-PAGE. Lane 1, aprotinin 6.5 kDa; Lane 10, pMAL-c2g induced with same IPTG to show the size of cytoplasmic MBP.</p>
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    <img src="https://static.igem.org/mediawiki/2020/6/6f/T--Fudan--2020-10-27-gel2.jpg" alt="1027 gel2" />
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    <h6>Figure 2. IPTG induced signal peptide fused CaAP</h6>
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    <p>Lane 4 and 5, bacteria DH5a with PhoA-CaAP, culture to OD 0.6; (lane 5) was induced with 1mM IPTG for 6 hours. Then, the bacteria whole-cell samples were run on 17% SDS-PAGE. Lane 1, aprotinin 6.5 kDa.</p>
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</div>
  
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  <h2>1. Five signal peptide for secretion</h2>
 
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    <p>We have constructed plasmids using CaAP or MBP(Maltose Binding Protein, a protein that is expressed in the cytoplasm of <i>E.coli</i>) with five signal peptides (BBa_K3606031~BBa_K3606035). We plan to test their amount of secretion and production in the same period. We have proven that OmpA, one of our signal peptides, can guide MBP to secret. We hypothesis the difference between each signal peptide is significant.</p>
 
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   <h2>2. 14 promoters for different strength expression</h2>
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   <h2>14 promoters for different strength expression</h2>
 
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     <img src="https://static.igem.org/mediawiki/2020/5/55/T--Fudan--promoters_3mL_12h.jpg" alt="Figure 1. Measurement of bacteria incubated in 3mL medium for 12h" />
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     <img src="https://static.igem.org/mediawiki/2020/5/55/T--Fudan--promoters_3mL_12h.jpg" alt="3mL medium for 12h" />
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     <p>Figure 1. Measurement of bacteria incubated in 3mL medium for 12h</p>
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     <h6>Figure 3. Bacteria with P1-P14 driven RFP incubated in 3mL medium for 12h</h6>
 
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     <img src="https://static.igem.org/mediawiki/2020/4/47/T--Fudan--promoters_3mL_24h.jpg"alt="Figure 2. Measurement of bacteria incubated in 3mL medium for 24h" />
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     <img src="https://static.igem.org/mediawiki/2020/4/47/T--Fudan--promoters_3mL_24h.jpg" alt="3mL medium for 24h" />
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     <p>Figure 2. Measurement of bacteria incubated in 3mL medium for 24h</p>
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     <h6>Figure 4. Bacteria with P1-P14 driven RFP incubated in 3mL medium for 24h</h6>
 
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     <p>We have constructed plasmids using RFP with 14 promoters for different strength expression (BBa_K3606045~BBa_K3606057) and have tested their fluorescence intensity. From the form above, the difference between each promoter is significant. </p>
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  <img src="https://static.igem.org/mediawiki/2020/0/09/T--Fudan--2020108-026.jpg" alt="mcbE F G" />
 +
  <h6>Figure 5. Ni-NTA bound CaAP</h6>
 +
     <p>Red dot, aprotinin 6.5 kDa, 16% SDS-PAGE. Lane 1, P3 driven CaAP;
 +
Lane 2, P4 driven CaAP;
 +
Lane 3, P5 driven CaAP;
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Lane 4, P6 driven CaAP;
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Lane 5, P7 driven CaAP;
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Lane 6, P8 driven CaAP;
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Lane 7, pGEX-6P1 (no 6xHis);
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Lane 8, P11 driven CaAP;
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Lane 9, P12 driven CaAP. For each sample, 3 OD bacteria were sonicated to lysis. And, 50 uL Ni-NTA beads (50% slurry) were mixed with cleared lysate for binding. Beads were washed in Buffer A, and then half of the beads were boiled in 100 uL with 1x SDS sample buffer, 10 uL was loaded to the lane.</p>
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<div class="column half_size">
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    <p><br/><br/>Using obtained P1-P14 driven RFP, we have constructed constructs expressing CaAP with 14 promoters for different strength expression (BBa_K3606045~BBa_K3606057). Those bacteria were grew to OD 1.5, broken up, and Ni-NTA beads were added into cleared lysate. A quick pull-down show clear bands of protein on the gel, which were bound to Ni-NTA beads, very likely are CaAP!</p>
 
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   <h2>3. IPTG inducible promoter driven mcbABCDEFG</h2>
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   <h2>IPTG inducible promoter driven mcbABCDEFG</h2>
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     <p>First, to affirm the system could work properly, we started from the very beginning by testing the expression of each gene in the cluster as well as of the polycistrons in each part. We constructed 10 plasmids(with pGEX backbone adapted to perform high level of expression), containing mcbA/ mcbB/ mcbC/ mcbD/ mcbE/ mcbF/ mcbG/ mcbABCD/ mcbBCD/ mcbBC respectively, transformed them into <i>E.coli</i> BL21, then purified the protein products with his-tag via immobilized metal affinity chromatography to check if the basic protein-coding could all be realized correctly as the basis of our design for the system. We confirmed that every part can express successfully due to the correct bands on the SDS-PAGE electrophoresis gel maps. </p>
+
     <p>First, to affirm the system could work properly, we started from the very beginning by testing the expression of each gene in the cluster as well as of the polycistrons in each part. We constructed 10 plasmids (with pGEX backbone adapted to perform high level of expression), containing mcbA/ mcbB/ mcbC/ mcbD/ mcbE/ mcbF/ mcbG/ mcbABCD/ mcbBCD/ mcbBC respectively, transformed them into <i>E. coli</i> BL21, then induced with IPTG. We confirmed that every part can express successfully due to the correct bands on the SDS-PAGE electrophoresis gel maps. </p>
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  </div>
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<div class="column half_size">
 +
  <img src="https://static.igem.org/mediawiki/2020/3/31/T--Fudan--2020108-027.jpg" alt="mcbE F G" />
 +
  <h6>Figure 6. IPTG induced mcbE mcbF mcbG</h6>
 +
    <p>Red dot, aprotinin 6.5 kDa; lane 2 vs 1, mcbE induced by IPTG; lane 4 vs 3, mcbF induced by IPTG; lane 6 vs 5, mcbG induced by IPTG. Bacteria were grown to OD 0.6, induced with or without 1 mM IPTG for 5 hours. Whole-cell samples were run. Yellow dots were placed to show induced bands in lane 2,4,6.</p>
 
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Revision as of 03:09, 28 October 2020
 
parts collection

Our part collection offers a complete array of parts for further researchers to develop other probiotic secreted proteins/peptides.

In the collection, we have secreted mcbABCD to help its colonization in the gut, and secreted CaAP to facilitate calcium uptake by gut cells. We test all parts used in these two circles: 5 signal peptide for secretion, 14 promoters for different strength expression (previously publish and available in addgene.org but not in the Registry), IPTG inducible promoter driven various protein coding sequences (confirmed individually before assembled together), and quorum-sensing components. A file contains those parts could be downloaded (updated on 2020-10-27).

We not only provide sequences and references for the parts, but also experimental results and procedures. We compete for both the Gold Medal criterion #7 and the Best Part Collection prize with this page.

Five signal peptide for secretion

We have constructed plasmids using CaAP or MBP (Maltose Binding Protein, a protein that is expressed in the cytoplasm of E.coli) with five signal peptides (CaAP fused with signal peptides: BBa_K3606031~BBa_K3606035; MBP fused: BBa_K3606037~BBa_K3606041). We have quickly test several of them: the total production and relative amount being secreted. We show that OmpA, one of our signal peptides, could lead MBP to secret. Our results consist with the literature that for a specfic protein all possible signal peptides should be test for best results.

1027 gel3
Figure 1. TCA precipated culture media (lane 8/with 9/no IPTG OmpA-MBP)

Lane 8 and 9, bacteria DH5a with OmpA-MBP, culture to OD 0.6 and induced with 1mM IPTG for 6 hours. Then, the culture media was collected and precipated with TCA. Samples were run on 17% SDS-PAGE. Lane 1, aprotinin 6.5 kDa; Lane 10, pMAL-c2g induced with same IPTG to show the size of cytoplasmic MBP.

1027 gel2
Figure 2. IPTG induced signal peptide fused CaAP

Lane 4 and 5, bacteria DH5a with PhoA-CaAP, culture to OD 0.6; (lane 5) was induced with 1mM IPTG for 6 hours. Then, the bacteria whole-cell samples were run on 17% SDS-PAGE. Lane 1, aprotinin 6.5 kDa.

14 promoters for different strength expression

3mL medium for 12h
Figure 3. Bacteria with P1-P14 driven RFP incubated in 3mL medium for 12h
3mL medium for 24h
Figure 4. Bacteria with P1-P14 driven RFP incubated in 3mL medium for 24h
mcbE F G
Figure 5. Ni-NTA bound CaAP

Red dot, aprotinin 6.5 kDa, 16% SDS-PAGE. Lane 1, P3 driven CaAP; Lane 2, P4 driven CaAP; Lane 3, P5 driven CaAP; Lane 4, P6 driven CaAP; Lane 5, P7 driven CaAP; Lane 6, P8 driven CaAP; Lane 7, pGEX-6P1 (no 6xHis); Lane 8, P11 driven CaAP; Lane 9, P12 driven CaAP. For each sample, 3 OD bacteria were sonicated to lysis. And, 50 uL Ni-NTA beads (50% slurry) were mixed with cleared lysate for binding. Beads were washed in Buffer A, and then half of the beads were boiled in 100 uL with 1x SDS sample buffer, 10 uL was loaded to the lane.



Using obtained P1-P14 driven RFP, we have constructed constructs expressing CaAP with 14 promoters for different strength expression (BBa_K3606045~BBa_K3606057). Those bacteria were grew to OD 1.5, broken up, and Ni-NTA beads were added into cleared lysate. A quick pull-down show clear bands of protein on the gel, which were bound to Ni-NTA beads, very likely are CaAP!

IPTG inducible promoter driven mcbABCDEFG

First, to affirm the system could work properly, we started from the very beginning by testing the expression of each gene in the cluster as well as of the polycistrons in each part. We constructed 10 plasmids (with pGEX backbone adapted to perform high level of expression), containing mcbA/ mcbB/ mcbC/ mcbD/ mcbE/ mcbF/ mcbG/ mcbABCD/ mcbBCD/ mcbBC respectively, transformed them into E. coli BL21, then induced with IPTG. We confirmed that every part can express successfully due to the correct bands on the SDS-PAGE electrophoresis gel maps.

mcbE F G
Figure 6. IPTG induced mcbE mcbF mcbG

Red dot, aprotinin 6.5 kDa; lane 2 vs 1, mcbE induced by IPTG; lane 4 vs 3, mcbF induced by IPTG; lane 6 vs 5, mcbG induced by IPTG. Bacteria were grown to OD 0.6, induced with or without 1 mM IPTG for 5 hours. Whole-cell samples were run. Yellow dots were placed to show induced bands in lane 2,4,6.

Signature: Yuanyuan