Difference between revisions of "Part:BBa K2669002:Experience"
m
 
(48 intermediate revisions by 3 users not shown)
Line 1: Line 1:
−
=== <p>Transformation results</p> ===
+
__NOTOC__
 +
===Applications of BBa_K2669002 ===
  
−
<html>
+
<br>
−
<img src="https://static.igem.org/mediawiki/parts/9/9b/Transformation_IDT_IDT_Colonies.jpeg" width="100%" height="100%">
+
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>.
−
<p><b>Figure 1:</b>Transformation plate of the IDT optimized AmilCP sequence. The circled colonies represent the colonies used in the stability assay.</p>
+
<br>
−
 
+
<br>
−
<img src="https://static.igem.org/mediawiki/parts/5/58/Transformation_Original_IDT-Colonies.jpeg" width="100%" height="100%">
+
https://static.igem.org/mediawiki/parts/thumb/f/f5/T--Guelph--pTA-tetnotet.jpg/216px-T--Guelph--pTA-tetnotet.jpg
−
<p><b>Figure 2:</b>Transformation plate of the original AmilCP sequence. The circled colonies represent the colonies used in the stability assay.</p>
+
<br>
−
</html>
+
<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).
−
 
+
<br>
−
=== <p>Stability assay</p> ===
+
<br>
−
<p>Stability assay through liquid experiment was performed with all three plasmids 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 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 protein. </p>
+
https://static.igem.org/mediawiki/parts/thumb/2/29/T--Guelph--pTAtetnotetspun.jpg/207px-T--Guelph--pTAtetnotetspun.jpg
−
<html>
+
<br>
−
<img src="https://static.igem.org/mediawiki/parts/c/cf/10_Gen_IDTbb.jpeg" width="100%" height="100%">
+
<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).
−
<p><b>Figure 3:</b> The result after 10 generations. The upper row represent 10 different colonies of the IDT optimized sequence of AmiLCP. The lower row represents the original AmilCP sequence. </p>
+
<br>
−
</html>
+
<br>
−
 
+
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.
−
=== <p>Interpretation</p> ===
+
<br>
−
<p> Already after 10 generations it was clearly visible that more IDT optimized colonies had the strongest color compared to the original amilCP colonies. This indicates that the stability of the IDT optimized AmiLCP is greater than for the original AmilCP sequence.
+
<br>
−
 
+
https://2019.igem.org/wiki/images/thumb/f/f5/T--Guelph--AmilCPtemexpress.jpg/800px-T--Guelph--AmilCPtemexpress.jpg
−
{| class="wikitable" style="margin-left: auto; margin-right: auto; border: none;
+
<br>
−
|+ Cells left-aligned, table centered
+
<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.
−
|-
+
<br>
−
! Generation
+
−
! IDT Optimized AmilCP Colony 1
+
−
! IDT Optimized AmilCP Colony 2
+
−
! IDT Optimized AmilCP Colony 3
+
−
! IDT Optimized AmilCP Colony 4
+
−
! IDT Optimized AmilCP Colony 5
+
−
! IDT Optimized AmilCP Colony 6
+
−
! IDT Optimized AmilCP Colony 7
+
−
! IDT Optimized AmilCP Colony 8
+
−
! IDT Optimized AmilCP Colony 9
+
−
! IDT Optimized AmilCP Colony 10
+
−
|-
+
−
! 10
+
−
| +++
+
−
| ++
+
−
| +++
+
−
| +++
+
−
| ++
+
−
| ++
+
−
| +++
+
−
| +++
+
−
| +++
+
−
| +
+
−
|}
+
−
 
+
−
<html>
+
−
<p><b>Table 1:</b> Interpretation of color expression of the IDT optimised AmilCP sequence after 10 generations of growth. +++ represents strong color, + represents weak color, - represents no color. </p>
+
−
</html>
+
−
 
+
−
 
+
−
{| class="wikitable" style="margin-left: auto; margin-right: auto; border: none;
+
−
|+ Cells left-aligned, table centered
+
−
|-
+
−
! Generation
+
−
! Original AmilCP Colony 1
+
−
! Original AmilCP Colony 2
+
−
! Original AmilCP Colony 3
+
−
! Original AmilCP Colony 4
+
−
! Original AmilCP Colony 5
+
−
! Original AmilCP Colony 6
+
−
! Original AmilCP Colony 7
+
−
! Original AmilCP Colony 8
+
−
! Original AmilCP Colony 9
+
−
! Original AmilCP Colony 10
+
−
|-
+
−
! 10
+
−
| +++
+
−
| +++
+
−
| +++
+
−
| +++
+
−
| +++
+
−
| +++
+
−
| +++
+
−
| +++
+
−
| +++
+
−
| +++
+
−
|}
+
−
 
+
−
<html>
+
−
<p><b>Table 2:</b> Interpretation of color expression of the original AmilCP sequence after 10 generations of growth. +++ represents strong color, + represents weak color, - represents no color. </p>
+
−
</html>
+
−
 
+
−
===Applications of BBa_K2669002===
+
  
 
===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

UNIQ2467a859e14cae8f-partinfo-00000000-QINU UNIQ2467a859e14cae8f-partinfo-00000001-QINU