Difference between revisions of "Part:BBa K2669002:Experience"

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<p> '''Note''' that the results were obtained with the plasmid design represented in the composite part: [https://parts.igem.org/Part:BBa_K2669003 IDT optimized AmilCP sequence]</p>
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===Applications of BBa_K2669002 ===
  
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=== <p>Transformation results</p> ===
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The construct [[BBa_K3189015]] containing the chromoprotein amilCP ([[BBa_K2669002]]) under the control of [[BBa_K3189001]]. When the system is induced with 100 ng/mL of tetracycline, a dark blue colour is produced (Figure 4 and Figure 5). This shows [[BBa_K3189001]] is able to function with different reporter proteins other than just <i>gfp</i>.
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<img src="https://static.igem.org/mediawiki/2018/7/79/T--Uppsala--T--Uppsala--Transformation_IDT_IDT-Colonies.jpeg" width="100%" height="100%">
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<p><b>Figure 1:</b> Transformation plate of colonies with the incorporated plasmid containing the IDT optimized original native AmilCP (BBa_K592009) sequence in the IDT supplied backbone. Colonies retrieved for the stability assay are circled.</p>
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https://static.igem.org/mediawiki/parts/thumb/f/f5/T--Guelph--pTA-tetnotet.jpg/216px-T--Guelph--pTA-tetnotet.jpg
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<img src="https://static.igem.org/mediawiki/2018/2/29/T--Uppsala--Transformation_Original_IDTbb-Colonies.jpeg" width="100%" height="100%">
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<b>Figure 1: [[BBa_K2669002]] under the control of [[BBa_K3189001]].</b> amilCP expression being induced using 100 ng/mL tetracycline (left) and uninduced (right).
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<p><b>Figure 2:</b> Transformation plate of colonies with the incorporated plasmid containing the original native AmilCP (BBa_K592009) sequence in the IDT supplied backbone. Colonies retrieved for the assay are circled.</p>
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https://static.igem.org/mediawiki/parts/thumb/2/29/T--Guelph--pTAtetnotetspun.jpg/207px-T--Guelph--pTAtetnotetspun.jpg
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=== <p>Stability assay</p> ===
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<p>Stability assay through growth in liquid culture was performed with both inserts in the IDT supplied backbone. 1 mL of LB with ampicillin (25 ug/ml) was inoculated with one single colony in eppendorf tubes in 10 replicates for each part. In order to allow 10 generations of growth, a 1:1000 dilution was made of the overnight culture and incubated for 24 h. The colour comparison was done through visualization of centrifuged cultures and by analyzing the color intensity we could deduce the stability of the chromoprotein-encoding plasmid. </p>
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<b>Figure 2: Pellets of cells of [[BBa_K2669002]] under the control of [[BBa_K3189001]] induced and uninduced with tetracycline.</b> Pellet of cells induced with 100 ng/mL tetracycline (left) and pellet of cells uninduced (right).
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<img src="https://static.igem.org/mediawiki/parts/e/e7/T--Uppsala--10_Gen_IDTbb_ORI_IDT.jpeg" width="100%" height="100%">
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<p><b>Figure 3:</b> The result after 10 generations. The upper row represents 10 different colonies of the cells with the plasmid containing the IDT optimized sequence of AmilCP. The lower row represents the ones with the original native AmilCP (BBa_K592009) sequence incorporated in the plasmid. </p>
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This year iGEM Guelph was working with BBa_K2669002 as a reporter protein. When working with this chromoprotein in <i>E.coli</i> BL21(DE3) under the control of [[BBa_K3189001]], we found that stronger colour was produced at lower temperatures compared to higher temperatures. In Figure 2 pigment intensity was compared between two temperatures, 25°C and 37°C. It is clear form these images that at 25°C the colonies have a darker colour compared to colonies grown at 37°C. This is evidence that AmilCP produced from [[BBa_K2669002]] is more stable at 25°C compared to 37°C thus allowing it to build up in the cells longer before degradation resulting in darker pigmentation of the colonies.
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=== <p>Interpretation</p> ===
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https://2019.igem.org/wiki/images/thumb/f/f5/T--Guelph--AmilCPtemexpress.jpg/800px-T--Guelph--AmilCPtemexpress.jpg
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<p> Already after 10 generations it was clearly visible that the color intensity was better distributed in the cells containing the plasmid with the IDT optimized AmilCP sequence, since all of the colonies had a large color intensity while the cells with the plasmid containing the original AmilCP sequence had a larger variation in color intensity. This indicates that the stability of the cells containing the plasmid with the IDT optimized AmilCP sequence is greater than those with the original AmilCP sequence. 
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<b>Figure 3: BBa_K2669002 expression at 25°C and 37°C.</b> Expression was carried out in <i>E.coli</i> BL21(DE3) on LB agar supplemented with 100 ug/mL ampicillin, 100 mg/mL tryptophan, 1mM IPTG, and 50 ng/mL tetracycline.
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<p><b>Table 1:</b> Interpretation of color expression of the cells containing the original AmilCP sequence or the IDT optimized AmilCP sequence after 10 generations of growth. ++ represents strong color, + represents weak color and - represents no color. </p>
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{| class="wikitable" style="margin-left: auto; margin-right: auto; border: none;
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|-
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! style="background: #98CC9A;" | Generation
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! style="background: #98CC9A;" | Original AmilCP Colony 1
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! style="background: #98CC9A;" | Original AmilCP Colony 2
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! style="background: #98CC9A;" | Original AmilCP Colony 3
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! style="background: #98CC9A;" | Original AmilCP Colony 4
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! style="background: #98CC9A;" | Original AmilCP Colony 5
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! style="background: #98CC9A;" | Original AmilCP Colony 6
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! style="background: #98CC9A;" | Original AmilCP Colony 7
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! style="background: #98CC9A;" | Original AmilCP Colony 8
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! style="background: #98CC9A;" | Original AmilCP Colony 9
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! style="background: #98CC9A;" | Original AmilCP Colony 10
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! style="background: #98CC9A;" | 10
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| ++
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| +
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| ++
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| ++
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| +
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| +
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| ++
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| ++
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| ++
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! style="background: #98CC9A;" | Generation
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! style="background: #98CC9A;" | IDT Optimized AmilCP Colony 1
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! style="background: #98CC9A;" | IDT Optimized AmilCP Colony 2
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! style="background: #98CC9A;" | IDT Optimized AmilCP Colony 3
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! style="background: #98CC9A;" | IDT Optimized AmilCP Colony 4
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! style="background: #98CC9A;" | IDT Optimized AmilCP Colony 5
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! style="background: #98CC9A;" | IDT Optimized AmilCP Colony 6
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! style="background: #98CC9A;" | IDT Optimized AmilCP Colony 7
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! style="background: #98CC9A;" | IDT Optimized AmilCP Colony 8
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! style="background: #98CC9A;" | IDT Optimized AmilCP Colony 9
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! style="background: #98CC9A;" | IDT Optimized AmilCP Colony 10
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! style="background: #98CC9A;" | 10
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| ++
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| ++
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| ++
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===Applications ===
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AmilCP can be used as a quantitative reporter.
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===User Reviews===
 
===User Reviews===

Latest revision as of 01:50, 22 October 2019

Applications of BBa_K2669002


The construct BBa_K3189015 containing the chromoprotein amilCP (BBa_K2669002) under the control of BBa_K3189001. When the system is induced with 100 ng/mL of tetracycline, a dark blue colour is produced (Figure 4 and Figure 5). This shows BBa_K3189001 is able to function with different reporter proteins other than just gfp.

216px-T--Guelph--pTA-tetnotet.jpg
Figure 1: BBa_K2669002 under the control of BBa_K3189001. amilCP expression being induced using 100 ng/mL tetracycline (left) and uninduced (right).

207px-T--Guelph--pTAtetnotetspun.jpg
Figure 2: Pellets of cells of BBa_K2669002 under the control of BBa_K3189001 induced and uninduced with tetracycline. Pellet of cells induced with 100 ng/mL tetracycline (left) and pellet of cells uninduced (right).

This year iGEM Guelph was working with BBa_K2669002 as a reporter protein. When working with this chromoprotein in E.coli BL21(DE3) under the control of BBa_K3189001, we found that stronger colour was produced at lower temperatures compared to higher temperatures. In Figure 2 pigment intensity was compared between two temperatures, 25°C and 37°C. It is clear form these images that at 25°C the colonies have a darker colour compared to colonies grown at 37°C. This is evidence that AmilCP produced from BBa_K2669002 is more stable at 25°C compared to 37°C thus allowing it to build up in the cells longer before degradation resulting in darker pigmentation of the colonies.

800px-T--Guelph--AmilCPtemexpress.jpg
Figure 3: BBa_K2669002 expression at 25°C and 37°C. Expression was carried out in E.coli BL21(DE3) on LB agar supplemented with 100 ug/mL ampicillin, 100 mg/mL tryptophan, 1mM IPTG, and 50 ng/mL tetracycline.

User Reviews

UNIQ5fb0b5ab3afba26a-partinfo-00000000-QINU UNIQ5fb0b5ab3afba26a-partinfo-00000001-QINU