Team:SCU China/SCU-Results

From 2013.igem.org

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-
We assemblied BBa_K1087004 with mRFP to create [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087015 BBa_K1087015], band we assembled [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087015 BBa_K1087015] with RiboKey BBa_K145215 to create [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087021 BBa_K1087021].
+
We assembled BBa_K1087004 with mRFP to create [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087015 BBa_K1087015], band we assembled [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087015 BBa_K1087015] with RiboKey BBa_K145215 to create [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087021 BBa_K1087021].
[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087015 BBa_K1087015]    [[File:SCU-r1.png]]
[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087015 BBa_K1087015]    [[File:SCU-r1.png]]
Line 47: Line 47:
 
 
-
We transformed [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087015 BBa_K1087015] and [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087021 BBa_K1087021] into ''E. coli'' DH5, respectively. And we used Ptet+mRFP1 (BBa_I13521) as positive control, [[App:app:ds:irrelevant|irrelevant]] BBa_J61046 with no [[App:app:ds:fluorescence|fluorescence]] gene as negative control.
+
We transformed [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087015 BBa_K1087015] and [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087021 BBa_K1087021] into ''E. coli'' DH5, respectively. And we used Ptet+mRFP1 (BBa_I13521) as positive control, irrelevant BBa_J61046 with no fluorescence gene as negative control.
 
 
Line 57: Line 57:
 
 
-
We used fluorescence microscope to observe the [[App:app:ds:fluorescence|fluorescence]] at 16h, and we also used Thermo Varioskan Flash to quantitatively measure the [[App:app:ds:fluorescence|fluorescence]] intensity at 16h, 18h, and 19h, respectively. 150ul culture fluid was added into each well of the 96-well black plate to measure the [[App:app:ds:fluorescence|fluorescence]] intensity, and OD600 were measured using spectrophotometer at the same time one by one to quantify the concentration of bacteria.
+
We used fluorescence microscope to observe the fluorescence at 16h, and we also used Thermo Varioskan Flash to quantitatively measure the fluorescence intensity at 16h, 18h, and 19h, respectively. 150ul culture fluid was added into each well of the 96-well black plate to measure the fluorescence intensity, and OD600 were measured using spectrophotometer at the same time one by one to quantify the concentration of bacteria.
-
 
+
-
Each sample had three duplicates when cultured. And the fluorescence value was [[App:app:ds:handle|handle]]d through [[App:app:ds:mathematical|mathematical]][[App:app:ds:method|method]]
+
 +
Each sample had three duplicates when cultured. And the fluorescence value was handled through mathematical method.
The fluorescence intensity of above transformants was divided by the OD600 value and minus that of negative control.
The fluorescence intensity of above transformants was divided by the OD600 value and minus that of negative control.
 
 
 +
Data  Qualitative representation of  Riboregulator 1, color change with time revealed the expression level  of corresponding parts.
 +
 +
 +
{| border=1px; cellspacing="0" cellpadding="0"
 +
|-
 +
| style="width: 84px" |
 +
 
 +
 +
| style="width: 84px" |
 +
13h
 +
 +
| style="width: 84px" |
 +
14.5h
 +
 +
| style="width: 84px" |
 +
16h
 +
 +
| style="width: 84px" |
 +
17.5
 +
 +
| style="width: 84px" |
 +
19h
 +
 +
| style="width: 84px" |
 +
20.5h
 +
 +
|-
 +
| style="width: 84px" |
 +
5H1
 +
 +
| style="width: 84px" |
 +
white
 +
 +
| style="width: 84px" |
 +
light pink
 +
 +
| style="width: 84px" |
 +
pink
 +
 +
| style="width: 84px" |
 +
pink
 +
 +
| style="width: 84px" |
 +
pink
 +
 +
| style="width: 84px" |
 +
rose red
 +
 +
|-
 +
| style="width: 84px" |
 +
5H2
 +
 +
| style="width: 84px" |
 +
white
 +
 +
| style="width: 84px" |
 +
light pink
 +
 +
| style="width: 84px" |
 +
pink
 +
 +
| style="width: 84px" |
 +
pink
 +
 +
| style="width: 84px" |
 +
pink
 +
 +
| style="width: 84px" |
 +
rose red
 +
 +
|-
 +
| style="width: 84px; height: 27px" |
 +
5H3
 +
 +
| style="width: 84px; height: 27px" |
 +
white
 +
 +
| style="width: 84px; height: 27px" |
 +
light pink
 +
 +
| style="width: 84px; height: 27px" |
 +
pink
 +
 +
| style="width: 84px; height: 27px" |
 +
pink
 +
 +
| style="width: 84px; height: 27px" |
 +
pink
 +
 +
| style="width: 84px; height: 27px" |
 +
rose red
 +
 +
|}
 +
 +
 +
 
 +
 +
[[File:SCU-rr1.png]] 
 +
 +
[[File:SCU-rr_(2).png]] 
 +
 +
[[File:SCU-rr_(3).png]] 
 +
 +
 
 +
 +
[[File:SCU-rr_(4).png]] 
 +
 +
[[File:SCU-rr_(5).png]] 
 +
 +
[[File:SCU-rr_(6).png]] 
 +
[[File:SCU-r4.png]] 
[[File:SCU-r4.png]] 
-
Figure 1.  [[App:app:ds:fluorescence|fluorescence]] observation using [[App:app:ds:fluorescence|fluorescence]] microscope.
+
Figure 1.  fluorescence observation using fluorescence microscope.
A. Negative control: Coletit DH5αtransformed with B Ba_J61046 in PSB1X3.     
A. Negative control: Coletit DH5αtransformed with B Ba_J61046 in PSB1X3.     
Line 81: Line 191:
[[File:SCU-r5.png]] 
[[File:SCU-r5.png]] 
-
Figure2.  quantitatively measurement of the [[App:app:ds:fluorescence|fluorescence]] intensity.
+
Figure2.  quantitatively measurement of the fluorescence intensity.
Red: Coletit DH5αtransformed with [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087015 BBa_K1087015].
Red: Coletit DH5αtransformed with [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087015 BBa_K1087015].
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{| border="1" cellspacing="0" cellpadding="0"
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{| border=1px; cellspacing="0" cellpadding="0"
|-
|-
| style="width: 72px; height: 54px" |  
| style="width: 72px; height: 54px" |  
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[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087022 BBa_K108702]3 [[File:SCU-r8.png]]
[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087022 BBa_K108702]3 [[File:SCU-r8.png]]
-
We transformed [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087018 BBa_K1087018], [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087022 BBa_K1087022], and [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087022 BBa_K108702]3 into ''E. coli'' DH5α, respectively. And we used [[App:app:ds:irrelevant|irrelevant]] BBa_J61046 with no [[App:app:ds:fluorescence|fluorescence]] gene as negative control. The transformed cells were cultured separately in the same condition and measured [[App:app:ds:fluorescence|fluorescence]] intensity at same time.
+
We transformed [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087018 BBa_K1087018], [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087022 BBa_K1087022], and [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087022 BBa_K108702]3 into ''E. coli'' DH5α, respectively. And we used irrelevant BBa    _J61046 with no fluorescence gene as negative control. The transformed cells were cultured separately in the same condition and measured fluorescence intensity at same time.
 
 
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The measurement was done mainly the same as result 1.
The measurement was done mainly the same as result 1.
 +
 +
 
 +
Data  Qualitative representation of  Riboregulator 2, color change with time revealed the expression level  of corresponding parts.
 +
 +
 +
{| border=1px; cellspacing="0" cellpadding="0"
 +
|-
 +
| style="width: 121px" |
 +
 
 +
 +
| style="width: 57px" |
 +
13h
 +
 +
| style="width: 85px" |
 +
14.5h
 +
 +
| style="width: 85px" |
 +
16h
 +
 +
| style="width: 85px" |
 +
17.5h
 +
 +
| style="width: 85px" |
 +
19h
 +
 +
| style="width: 94px" |
 +
20.5h
 +
 +
|-
 +
| style="width: 121px" |
 +
7H+22O 1
 +
 +
| style="width: 57px" |
 +
white
 +
 +
| style="width: 85px" |
 +
light yellow
 +
 +
| style="width: 85px" |
 +
light yellow
 +
 +
| style="width: 85px" |
 +
light yellow
 +
 +
| style="width: 85px" |
 +
light pink
 +
 +
| style="width: 94px" |
 +
light pink
 +
 +
|-
 +
| style="width: 121px" |
 +
7H+22O 2
 +
 +
| style="width: 57px" |
 +
white
 +
 +
| style="width: 85px" |
 +
light yellow
 +
 +
| style="width: 85px" |
 +
light yellow
 +
 +
| style="width: 85px" |
 +
light yellow
 +
 +
| style="width: 85px" |
 +
light pink
 +
 +
| style="width: 94px" |
 +
light pink
 +
 +
|-
 +
| style="width: 121px" |
 +
6I+3N+12G+23L+7H+22O 1
 +
 +
| style="width: 57px" |
 +
white
 +
 +
| style="width: 85px" |
 +
light yellow
 +
 +
| style="width: 85px" |
 +
light yellow
 +
 +
| style="width: 85px" |
 +
light yellow
 +
 +
| style="width: 85px" |
 +
light yellow
 +
 +
| style="width: 94px" |
 +
light yellow
 +
 +
|-
 +
| style="width: 121px" |
 +
6I+3N+12G+23L+7H+22O 2
 +
 +
| style="width: 57px" |
 +
white
 +
 +
| style="width: 85px" |
 +
light yellow
 +
 +
| style="width: 85px" |
 +
light yellow
 +
 +
| style="width: 85px" |
 +
light yellow
 +
 +
| style="width: 85px" |
 +
light yellow
 +
 +
| style="width: 94px" |
 +
light yellow
 +
 +
|-
 +
| style="width: 121px" |
 +
13J+6I+3N+12G+23L+7H+22O 1
 +
 +
| style="width: 57px" |
 +
white
 +
 +
| style="width: 85px" |
 +
light pink
 +
 +
| style="width: 85px" |
 +
light pink
 +
 +
| style="width: 85px" |
 +
light pink
 +
 +
| style="width: 85px" |
 +
 pink
 +
 +
| style="width: 94px" |
 +
rose red
 +
 +
|-
 +
| style="width: 121px" |
 +
13J+6I+3N+12G+23L+7H+22O 2
 +
 +
| style="width: 57px" |
 +
white
 +
 +
| style="width: 85px" |
 +
Light pink
 +
 +
| style="width: 85px" |
 +
Light pink
 +
 +
| style="width: 85px" |
 +
Light pink
 +
 +
| style="width: 85px" |
 +
pink
 +
 +
| style="width: 94px" |
 +
Rose red
 +
 +
|}
 +
 +
 +
 
 +
 +
 +
 
 +
 +
 +
 +
[[File:SCU-rr_(11).png]] 
 +
 +
[[File:SCU-rr_(12).png]] 
 +
 +
[[File:SCU-rr_(13).png]] 
 +
 +
[[File:SCU-rr_(14).png]] 
 +
 +
[[File:SCU-rr_(15).png]] 
 +
 +
[[File:SCU-rr_(16).png]] 
 
 
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[[File:SCU-r9.png]] 
[[File:SCU-r9.png]] 
-
Figure 1. Figure 1.  [[App:app:ds:fluorescence|fluorescence]] observation using [[App:app:ds:fluorescence|fluorescence]] microscope.
+
Figure 1. Figure 1.  fluorescence observation using fluorescence microscope.
A. Negative control: Ecoli DH5αtransformed with BBa_J61046 in PSB1X3.     
A. Negative control: Ecoli DH5αtransformed with BBa_J61046 in PSB1X3.     
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[[File:SCU-r10.png]] 
[[File:SCU-r10.png]] 
-
Figure2. Quantitatively measurement of the [[App:app:ds:fluorescence|fluorescence]] intensity.
+
Figure2. Quantitatively measurement of the fluorescence intensity.
Blue: ''E. coli'' DH5αtransformed with [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087015 BBa_K10870]23.
Blue: ''E. coli'' DH5αtransformed with [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087015 BBa_K10870]23.
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{| border="1" cellspacing="0" cellpadding="0"
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{| border=1px; cellspacing="0" cellpadding="0"
|-
|-
| style="width: 72px; height: 54px" |  
| style="width: 72px; height: 54px" |  
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[[File:SCU-r11.png]] 
[[File:SCU-r11.png]] 
-
Normally, we picked two colonies from the agar plate for [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087018 BBa_K108701]6, called A and B. We transformed [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087018 BBa_K108701]6A and B into ''E. coli'' DH5, respectively. And we use Ptet+mRFP1(BBa_I13521) as positive control, [[App:app:ds:irrelevant|irrelevant]] BBa_J61046 with no [[App:app:ds:fluorescence|fluorescence]] gene as negative control. The transformed cells were cultured separately in the same condition and measured [[App:app:ds:fluorescence|fluorescence]] intensity at same time.
+
Normally, we picked two colonies from the agar plate for [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087018 BBa_K108701]6, called A and B. We transformed [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087018 BBa_K108701]6A and B into ''E. coli'' DH5, respectively. And we use Ptet+mRFP1(BBa_I13521) as positive control, irrelevant BBa_J61046 with no fluorescence gene as negative control. The transformed cells were cultured separately in the same condition and measured fluorescence intensity at same time.
 
 
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The measurement was done mainly the same as result 1.
The measurement was done mainly the same as result 1.
 +
 +
 
 +
 +
 +
 +
 +
Data  Qualitative representation of  Riboregulator 3 , color change with time revealed the expression level  of corresponding parts.
 +
 +
 
 +
 +
 +
{| border=1px; cellspacing="0" cellpadding="0"
 +
|-
 +
| style="width: 98px" |
 +
 
 +
 +
| style="width: 98px" |
 +
13.5h
 +
 +
| style="width: 98px" |
 +
15h
 +
 +
| style="width: 98px" |
 +
16.5h
 +
 +
| style="width: 98px" |
 +
18h
 +
 +
|-
 +
| style="width: 98px" |
 +
2-1
 +
 +
| style="width: 98px" rowspan="2" |
 +
white
 +
 +
| style="width: 98px" rowspan="2" |
 +
white
 +
 +
| style="width: 98px" rowspan="2" |
 +
white
 +
 +
| style="width: 98px" rowspan="2" |
 +
Light pink
 +
 +
|-
 +
| style="width: 98px" |
 +
2-2
 +
 +
|-
 +
| style="width: 98px" |
 +
3-1
 +
 +
| style="width: 98px" rowspan="2" |
 +
Light pink
 +
 +
| style="width: 98px" rowspan="2" |
 +
pink
 +
 +
| style="width: 98px" rowspan="2" |
 +
Rose red
 +
 +
| style="width: 98px" rowspan="2" |
 +
Rose red
 +
 +
|-
 +
| style="width: 98px" |
 +
3-2
 +
 +
|-
 +
| style="width: 98px" |
 +
4-1
 +
 +
| style="width: 98px" rowspan="2" |
 +
white
 +
 +
| style="width: 98px" rowspan="2" |
 +
white
 +
 +
| style="width: 98px" rowspan="2" |
 +
Light pink
 +
 +
| style="width: 98px" rowspan="2" |
 +
pink
 +
 +
|-
 +
| style="width: 98px" |
 +
4-2
 +
 +
|}
 +
 +
 +
 
 +
 +
 
 +
[[File:SCU-rr_(7).png]] 
 +
 +
[[File:SCU-rr_(8).png]] 
 +
 +
[[File:SCU-rr_(9).png]] 
 +
 +
[[File:SCU-rr_(10).png]] 
 +
 +
 
 +
 +
 
 +
 +
 
 +
 +
 
 
 
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[[File:SCU-r12.png]] 
[[File:SCU-r12.png]] 
-
Figure1. [[App:app:ds:fluorescence|Fluorescence]] observation using [[App:app:ds:fluorescence|fluorescence]] microscope.
+
Figure1. fluorescence observation using fluorescence microscope.
A. Negative control: ''E. coli'' DH5αtransformed with BBa_J61046 in PSB1X3.     
A. Negative control: ''E. coli'' DH5αtransformed with BBa_J61046 in PSB1X3.     
Line 428: Line 828:
[[File:SCU-r13.png]] 
[[File:SCU-r13.png]] 
-
Figure2. Quantitatively measurement of the [[App:app:ds:fluorescence|fluorescence]] intensity.
+
Figure2. Quantitatively measurement of the fluorescence intensity.
Blue: ''E. coli'' DH5αtransformed with [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087018 BBa_K108701]6A.
Blue: ''E. coli'' DH5αtransformed with [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087018 BBa_K108701]6A.
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{| border="1" cellspacing="0" cellpadding="0"
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{| border=1px; cellspacing="0" cellpadding="0"
|-
|-
| style="width: 72px; height: 54px" |  
| style="width: 72px; height: 54px" |  
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|}
|}
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-
 
 
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|}
|}
 +
__NOEDITSECTION__

Latest revision as of 19:01, 13 October 2013

Animation

 


Contents

Result

Result 1 Test the function of Riboregulator

 

We assembled BBa_K1087004 with mRFP to create [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087015 BBa_K1087015], band we assembled [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087015 BBa_K1087015] with RiboKey BBa_K145215 to create [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087021 BBa_K1087021].

[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087015 BBa_K1087015]    SCU-r1.png

[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087021 BBa_K1087021]    SCU-r2.png

Positive control    SCU-r3.png

 

Negative control:  BBa_J61046

 

We transformed [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087015 BBa_K1087015] and [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087021 BBa_K1087021] into E. coli DH5, respectively. And we used Ptet+mRFP1 (BBa_I13521) as positive control, irrelevant BBa_J61046 with no fluorescence gene as negative control.

 

All of the constructs were expressed in high copy plasmid PSB1X3.

The transformed cells were cultured in 37℃, 200rpm.

 

We used fluorescence microscope to observe the fluorescence at 16h, and we also used Thermo Varioskan Flash to quantitatively measure the fluorescence intensity at 16h, 18h, and 19h, respectively. 150ul culture fluid was added into each well of the 96-well black plate to measure the fluorescence intensity, and OD600 were measured using spectrophotometer at the same time one by one to quantify the concentration of bacteria.

Each sample had three duplicates when cultured. And the fluorescence value was handled through mathematical method. The fluorescence intensity of above transformants was divided by the OD600 value and minus that of negative control.

  Data  Qualitative representation of  Riboregulator 1, color change with time revealed the expression level  of corresponding parts.


 

13h

14.5h

16h

17.5

19h

20.5h

5H1

white

light pink

pink

pink

pink

rose red

5H2

white

light pink

pink

pink

pink

rose red

5H3

white

light pink

pink

pink

pink

rose red


 

SCU-rr1.png 

SCU-rr (2).png 

SCU-rr (3).png 

 

SCU-rr (4).png 

SCU-rr (5).png 

SCU-rr (6).png 


SCU-r4.png 

Figure 1.  fluorescence observation using fluorescence microscope.

A. Negative control: Coletit DH5αtransformed with B Ba_J61046 in PSB1X3.     

B. Coletit DH5αtransformed with [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087015 BBa_K1087015].      

C. Coletit DH5αtransformed with [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087015 BBa_K10870]21.

D. Positive control: Coletit DH5αtransformed with B Ba_I13521.

 

SCU-r5.png 

Figure2.  quantitatively measurement of the fluorescence intensity.

Red: Coletit DH5αtransformed with [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087015 BBa_K1087015].

Blue: Coletit DH5αtransformed with [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087015 BBa_K10870]21.

Black: Positive control: Coletit DH5αtransformed with B Ba_I13521.

PS: the fluorescence intensity of above transformants was divided by the OD600 value and minus that of negative control.

 

 

According to above figures, it’s obvious that the ribolock BBa_K1087004 worked well, and its fluorescence intensity was only 2.6531% compared to positive control even at 19h, while that of lock & key ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087021 BBa_K1087021]) was 91.7138% at 19h. The results suggested that the key BBa_K145215 could hugely improve the expression level of the lock BBa_K1087004.

 

Data:


Fluorescence per OD

[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087015 BBa_K1087015]

[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087015 BBa_K10870]21

BBa_I13521

16h

0.089

6.8595

14.849

18h

0.486

16.011

18.363

19h

0.629

21.7435

23.708

percentage

[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087015 BBa_K1087015]

[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087015 BBa_K10870]21

BBa_I13521

16h

0.00599367

0.4619503

1

18h

0.026466264

0.871916354

1

19h

0.026531129

0.917137675

1


Result 2 Test the function of the Riboregulator and PhiR73 activator together

 

We assembled PO promoter with mRFP1 to create [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087018 BBa_K1087018]，so that we can test the basal expression level of PO promoter.

We also assembled “TetR promoter + lock3d” with phiR73 activator to create [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087007 BBa_K1087007]. Then, we assembly [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087007 BBa_K1087007] with [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087018 BBa_K1087018] to create [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087022 BBa_K1087022].

Finally, we assembled RiboKey BBa_K145215 with [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087022 BBa_K1087022] to create [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087022 BBa_K108702]3.

 

[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087018 BBa_K1087018]                             SCU-r6.png

 

[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087022 BBa_K1087022]            SCU-r7.png

 

[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087022 BBa_K108702]3 SCU-r8.png

We transformed [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087018 BBa_K1087018], [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087022 BBa_K1087022], and [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087022 BBa_K108702]3 into E. coli DH5α, respectively. And we used irrelevant BBa _J61046 with no fluorescence gene as negative control. The transformed cells were cultured separately in the same condition and measured fluorescence intensity at same time.

 

 

The measurement was done mainly the same as result 1.

  Data  Qualitative representation of  Riboregulator 2, color change with time revealed the expression level  of corresponding parts.


 

13h

14.5h

16h

17.5h

19h

20.5h

7H+22O 1

white

light yellow

light yellow

light yellow

light pink

light pink

7H+22O 2

white

light yellow

light yellow

light yellow

light pink

light pink

6I+3N+12G+23L+7H+22O 1

white

light yellow

light yellow

light yellow

light yellow

light yellow

6I+3N+12G+23L+7H+22O 2

white

light yellow

light yellow

light yellow

light yellow

light yellow

13J+6I+3N+12G+23L+7H+22O 1

white

light pink

light pink

light pink

 pink

rose red

13J+6I+3N+12G+23L+7H+22O 2

white

Light pink

Light pink

Light pink

pink

Rose red


 


 


SCU-rr (11).png 

SCU-rr (12).png 

SCU-rr (13).png 

SCU-rr (14).png 

SCU-rr (15).png 

SCU-rr (16).png 

 

SCU-r9.png 

Figure 1. Figure 1.  fluorescence observation using fluorescence microscope.

A. Negative control: Ecoli DH5αtransformed with BBa_J61046 in PSB1X3.     

B. E. coli DH5αtransformed with [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087018 BBa_K1087018].      

C. E. coli DH5αtransformed with [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087015 BBa_K10870]22.

D. E. coli DH5αtransformed with [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087015 BBa_K10870]23.

 

SCU-r10.png 

Figure2. Quantitatively measurement of the fluorescence intensity.

Blue: E. coli DH5αtransformed with [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087015 BBa_K10870]23.

Green: E. coli DH5αtransformed with [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087015 BBa_K10870]18.

Pink: E. coli DH5αtransformed with BBa_K108722.

PS: the fluorescence intensity of above transformants was divided by the OD600 value and minus that of negative control.

 

We could see clearly from above figures that the basal expression level of PO promoter was really low, and its strength could be improved about 11 times when induced by PhiR73 controlled by ribokey and lock.

Data:


Fluorescence per OD

[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087015 BBa_K10870]23

BBa_K108722

[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087015 BBa_K10870]18

16h

19.55141667

1.29

2.311

18h

49.09341667

1.5785

3.885

19h

66.099625

1.43575

5.043

fold

[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087015 BBa_K10870]23

BBa_K108722

[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087015 BBa_K10870]18

16h

8.460154334

0.558199913

1

18h

12.63665809

0.406306306

1

19h

13.1072

0.2847

1


 

 

 

 

 

 

Result 3 Test the function of Part BBa_K1087016 BBa_K1087016

SCU-r11.png 

Normally, we picked two colonies from the agar plate for [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087018 BBa_K108701]6, called A and B. We transformed [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087018 BBa_K108701]6A and B into E. coli DH5, respectively. And we use Ptet+mRFP1(BBa_I13521) as positive control, irrelevant BBa_J61046 with no fluorescence gene as negative control. The transformed cells were cultured separately in the same condition and measured fluorescence intensity at same time.

 

 

The measurement was done mainly the same as result 1.

 



Data  Qualitative representation of  Riboregulator 3 , color change with time revealed the expression level  of corresponding parts.

 


 

13.5h

15h

16.5h

18h

2-1

white

white

white

Light pink

2-2

3-1

Light pink

pink

Rose red

Rose red

3-2

4-1

white

white

Light pink

pink

4-2


 

  SCU-rr (7).png 

SCU-rr (8).png 

SCU-rr (9).png 

SCU-rr (10).png 

 

 

 

 

 

SCU-r12.png 

Figure1. fluorescence observation using fluorescence microscope.

A. Negative control: E. coli DH5αtransformed with BBa_J61046 in PSB1X3.     

B. E. coli DH5αtransformed with [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087018 BBa_K108701]6A.      

C. E. coli DH5αtransformed with [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087018 BBa_K108701]6B.

D. Positive control: E. coli DH5αtransformed with BBa_I13521.

 

 

 

SCU-r13.png 

Figure2. Quantitatively measurement of the fluorescence intensity.

Blue: E. coli DH5αtransformed with [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087018 BBa_K108701]6A.

Black: Positive control, E. coli DH5αtransformed with BBa_I13521.

Green: E. coli DH5αtransformed with [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087018 BBa_K108701]6B.

PS: the fluorescence intensity of above transformants was divided by the OD600 value and minus that of negative control.

 

From above figures, we could conclude that the Ptet and mRFP1 in [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087018 BBa_K108701]6 worked well. It was comparable to even stronger than the positive control BBa_I13521 in fluorescence intensity. For [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087018 BBa_K108701]6A, it expressed about twice fluorescence of BBa_I13521. Results for sequencing suggested that the first half of the Ptet was lost in [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087018 BBa_K108701]6A, which may account for its increase in strength.

 

Data:


Fluorescence per OD

[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087018 BBa_K108701]6B

[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087018 BBa_K108701]6A

BBa_I13521

16h

5.4215

19.23766667

14.849

18h

11.37075

36.49133333

18.363

19h

15.27725

47.6205

23.708

fold

[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087018 BBa_K108701]6B

[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1087018 BBa_K108701]6A

BBa_I13521

16h

0.365108762

1.295553011

1

18h

0.619220716

1.987220679

1

19h

0.644392188

2.00862578

1

 

 

Retrieved from "http://2013.igem.org/Team:SCU_China/SCU-Results"