Team:SUSTC-Shenzhen-B/RFC

From 2012.igem.org

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<br>
<br>
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Jiankui He, Shui Jing, Mengshi Zhang, Xianggeng Liu, Yao Chen, Boyu Qie, Liuxing
+
<p>Jiankui He, Shui Jing, Mengshi Zhang, Xianggeng Liu, Yao Chen, Boyu Qie, Liuxing
-
Shen Zishan Wu
+
Shen Zishan Wu</p>
-
26 September 2012
+
<p>26 September 2012</p>
-
1. Purpose
+
<p>1. Purpose</p>
-
The purpose of this RFC is to provide standard methodology for the measurement
+
<p>The purpose of this RFC is to provide standard methodology for the measurement
of the absolute strength of terminators in bacteria. Because we have characterized
of the absolute strength of terminators in bacteria. Because we have characterized
the performance of terminator in E.coli and used a simple equation model, it can be
the performance of terminator in E.coli and used a simple equation model, it can be
-
expressed in PoPS.
+
expressed in PoPS.</p>
-
2. Relation to other BBF RFCs
+
<p>2. Relation to other BBF RFCs</p>
-
This maybe doesn’t relate to any other BBF RFC.
+
<p>This maybe doesn’t relate to any other BBF RFC.</p>
-
3. Copyright Notice
+
<p>3. Copyright Notice</p>
-
Copyright (C) The BioBrick foundation(2012).All Right Reserved.
+
<p>Copyright (C) The BioBrick foundation(2012).All Right Reserved.</p>
-
4. Standard terminator efficiency measurement kit for use in E.coli.
+
<p>4. Standard terminator efficiency measurement kit for use in E.coli.</p>
-
The terminator efficiency measurement kit MUST include promoter, ribosome
+
<p>The terminator efficiency measurement kit MUST include promoter, ribosome
binding sites, reporter gene and vector backbone. Standard transcription terminator
binding sites, reporter gene and vector backbone. Standard transcription terminator
efficiency measurement kit MUST comprise of promoter BBa_R0040, ribosome
efficiency measurement kit MUST comprise of promoter BBa_R0040, ribosome
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comprise vector backbone pSB1A3, a high copy number plasmid carrying ampicillin
comprise vector backbone pSB1A3, a high copy number plasmid carrying ampicillin
resistance. This high copy number vector is chosen to facilitate growth of bacteria
resistance. This high copy number vector is chosen to facilitate growth of bacteria
-
step in the experiment
+
step in the experiment</p>
-
5. Design and construction of characterization devices.
+
<p>5. Design and construction of characterization devices.</p>
-
The fluorescence produced by the characterization devices are then measured using
+
<p>The fluorescence produced by the characterization devices are then measured using
-
flow cytometry to calculate the termination efficiency of the terminators.
+
flow cytometry to calculate the termination efficiency of the terminators.</p>
-
Experimental Group:
+
<p>Experimental Group:</p>
-
(Inputs are measured by RFP expression and outputs are measured by GFP
+
<p>(Inputs are measured by RFP expression and outputs are measured by GFP
-
expression.)
+
expression.)</p>
-
Control Group:
+
<p>Control Group:</p>
-
(calibrates RFP input to GFP output.)
+
<p>(calibrates RFP input to GFP output.)</p>
-
6. Experimental procedure
+
<p>6. Experimental procedure</p>
-
6.1 Media:
+
<p>6.1 Media:</p>
-
Lysogeny Broth (LB) liquid and solid media.
+
<p>Lysogeny Broth (LB) liquid and solid media.</p>
-
6.2 Protocol:
+
<p>6.2 Protocol:</p>
-
a. Plasmid pSB1A3 is chosen to be the vector that ligate GPF and RFP fragments.
+
<p>a. Plasmid pSB1A3 is chosen to be the vector that ligate GPF and RFP fragments.
To protect the structural integrity of the constructed plasmid, we need to
To protect the structural integrity of the constructed plasmid, we need to
mutate a restriction enzyme cutting site named Pst I to Afl II using PCR.
mutate a restriction enzyme cutting site named Pst I to Afl II using PCR.
-
Proper primer are designed for this purpose.
+
Proper primer are designed for this purpose.</p>
-
PtoA-F:5'-CCACCTGACGTCTAAGAAAC-3'
+
<p>PtoA-F:5'-CCACCTGACGTCTAAGAAAC-3'</p>
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PtoA-R:5'-ATGATCATCGCCGGCGAATTCAGGC-3'
+
<p>PtoA-R:5'-ATGATCATCGCCGGCGAATTCAGGC-3'</p>
-
b. Check whether the mutation of restriction cutting site is successful.
+
<p>b. Check whether the mutation of restriction cutting site is successful.</p>
-
c.
+
<p>c.Transform the plasmid to competent cell DH5αand incubate overnight.</p>
-
d. Pick up colonies, plasmid isolation, and digest to choose the positive
+
<p>d. Pick up colonies, plasmid isolation, and digest to choose the positive
-
one.
+
one.</p>
-
e. Amplify GFP & RFP. Do an electrophoresis for verification the PCR is
+
<p>e. Amplify GFP & RFP. Do an electrophoresis for verification the PCR is
-
successful.
+
successful.</p>
-
f.
+
<p>f.
Use specific restriction enzymes to digest plasmid mutant-psb1a3,GFP
Use specific restriction enzymes to digest plasmid mutant-psb1a3,GFP
and RFP to get sticky ends and purify the DNA fragment after the
and RFP to get sticky ends and purify the DNA fragment after the
-
Electrophoresis.
+
Electrophoresis.</p>
-
Digestion of plasmid mutant-pSB1A3 with Afl II and Not I;
+
<p>Digestion of plasmid mutant-pSB1A3 with Afl II and Not I;</p>
-
Digestion of PCR product GFP with Not I and Spe I;
+
<p>Digestion of PCR product GFP with Not I and Spe I;</p>
-
Digestion of PCR product RFP with Afl II and Spe I;
+
<p>Digestion of PCR product RFP with Afl II and Spe I;</p>
-
Put the tubes in 37℃ environment for 4-8 hours.
+
<p>Put the tubes in 37℃ environment for 4-8 hours.</p>
-
g. Ligation is needed to connect these 3 fragments together.
+
<p>g. Ligation is needed to connect these 3 fragments together.</p>
-
h.
+
<p>h.Transform the plasmid to competent cell DH5αand incubate overnight.</p>
-
i.Pick up colonies, plasmid isolation, and choose the positive one.
+
<p>i.Pick up colonies, plasmid isolation, and choose the positive one.</p>
-
Use restriction enzymes Xba I and Pst I to digest the constructed vector.
+
<p>j. Use restriction enzymes Xba I and Pst I to digest the constructed vector.</p>
-
j.
 
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k. Ligation is needed to terminators to be measured and the vector together.
+
<p>k. Ligation is needed to terminators to be measured and the vector together.</p>
-
Transform the plasmid to competent cell and incubate overnight.
+
Transform the plasmid to competent cell and incubate overnight.</p>
-
l.
+
<p>l. Transform the plasmid to competent cell DH5αand incubate overnight.</p>
-
Transform the plasmid to competent cell DH5αand incubate overnight.
+
<p>m. Test the fluorescent using the method of flow cytometry.</p>
-
Transform the plasmid to competent cell DH5αand incubate overnight.
+
<p>7. Parts used to create the terminator characterization.</p>
-
m. Test the fluorescent using the method of flow cytometry.
 
-
7. Parts used to create the terminator characterization.
+
<p>8. Calculate terminator efficiency</p>
 +
<p>TE=1-GE/GC</p>
 +
<p>TE: Terminator efficiency</p>
 +
<p>GE: Mean value of GFP from experimental group</p>
 +
<p>GC: Mean value of GFP from control group</p>
-
Left Part
+
<p>Under the ideal circumstances, the output of GFP and RFP should be almost the same and
-
BBa_E1010
+
-
BBa_E1010
+
-
BBa_E1010
+
-
BBa_E1010
+
-
BBa_E1010
+
-
BBa_E1010
+
-
BBa_E1010
+
-
BBa_E1010
+
-
BBa_E1010
+
-
 
+
-
8.
+
-
Calculate terminator efficiency
+
-
TE=1-GE/GC
+
-
TE: Terminator efficiency
+
-
GE: Mean value of GFP from experimental group
+
-
GC: Mean value of GFP from control group
+
-
 
+
-
Under the ideal circumstances, the output of GFP and RFP should be almost the same and
+
the value of TE should approach 0. If a strong terminator is placed into characterization
the value of TE should approach 0. If a strong terminator is placed into characterization
experimental group plasmid, the only output should be RFP and the value of TE should
experimental group plasmid, the only output should be RFP and the value of TE should
-
approach 1.
+
approach 1.</p>
-
9. Author’s contact information
+
<p>9. Author’s contact information</p>
-
Jiankui He:
+
<p>Jiankui He:
-
jiankuihe@gmail.com
+
jiankuihe@gmail.com</p>
-
Shui Jing:
+
<p>Shui Jing:
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betty.jingshui@gmail.com
+
betty.jingshui@gmail.com</p>
-
Mengshi Zhang: mengshi0928@gmail.com
+
<p>Mengshi Zhang: mengshi0928@gmail.com</p>
-
Xianggeng Liu: liuxianggeng@gmail.com
+
<p>Xianggeng Liu: liuxianggeng@gmail.com</p>
-
Yao Chen:
+
<p>Yao Chen:
-
chen.yy@live.com
+
chen.yy@live.com</p>
-
Boyu Qie:
+
<p>Boyu Qie:
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boyuqie@gmail.com
+
boyuqie@gmail.com</p>
-
Liuxing Shen: shenliuxing@gmail.com
+
<p>Liuxing Shen: shenliuxing@gmail.com</p>
-
Zishan Wu:
+
<p>Zishan Wu:
-
zishan.woo@gmail.com
+
zishan.woo@gmail.com</p>
-
10. References
+
<p>10. References</p>
-
[1] http://partsregistry.org/wiki/index.php/Part_Types:Measurement_Systems
+
<p>[1] http://partsregistry.org/wiki/index.php/Part_Types:Measurement_Systems</p>
-
[2] Haiyao Huang “Design and Characterization of Artificial Transcriptional
+
<p>[2] Haiyao Huang “Design and Characterization of Artificial Transcriptional
-
Terminators”.
+
Terminators”.</p>
-
[3] Lars Velten, Nao” Units for Promoter Measurement in Mammalian Cells”
+
<p>[3] Lars Velten, Nao” Units for Promoter Measurement in Mammalian Cells”</p>
-
Right Part
 
-
BBa_E0040
 
-
BBa_E0040
 
-
BBa_E0040
 
-
BBa_E0040
 
-
BBa_E0040
 
-
BBa_E0040
 
-
BBa_E0040
 
-
BBa_E0040
 
-
BBa_E0040
 
-
Description
 
-
RFP+BBa_K827086+GFP
 
-
RFP+BBa_K827087+GFP
 
-
RFP+BBa_K827088+GFP
 
-
RFP+BBa_K827089+GFP
 
-
RFP+BBa_K827090+GFP
 
-
RFP+BBa_K827091+GFP
 
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RFP+BBa_K827092+GFP
 
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RFP+BBa_K827093+GFP
 
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RFP+BBa_K827094+GFP
 
<div class="cleaner h40"></div>
<div class="cleaner h40"></div>
   
   

Revision as of 03:07, 27 September 2012

SUSTC iGEM Theme - Free CSS Template

SUSTC iGEM Theme - Free CSS Template

BBF RFC 90：The Measurement of rho-independent Transcription Terminator Efficiency.


Jiankui He, Shui Jing, Mengshi Zhang, Xianggeng Liu, Yao Chen, Boyu Qie, Liuxing Shen Zishan Wu

26 September 2012

1. Purpose

The purpose of this RFC is to provide standard methodology for the measurement of the absolute strength of terminators in bacteria. Because we have characterized the performance of terminator in E.coli and used a simple equation model, it can be expressed in PoPS.

2. Relation to other BBF RFCs

This maybe doesn’t relate to any other BBF RFC.

3. Copyright Notice

Copyright (C) The BioBrick foundation(2012).All Right Reserved.

4. Standard terminator efficiency measurement kit for use in E.coli.

The terminator efficiency measurement kit MUST include promoter, ribosome binding sites, reporter gene and vector backbone. Standard transcription terminator efficiency measurement kit MUST comprise of promoter BBa_R0040, ribosome binding site BBa_B0030, reporter gene, RFP BBa_E1010, GFP BBa_E0040. It SHOULD comprise vector backbone pSB1A3, a high copy number plasmid carrying ampicillin resistance. This high copy number vector is chosen to facilitate growth of bacteria step in the experiment

5. Design and construction of characterization devices.

The fluorescence produced by the characterization devices are then measured using flow cytometry to calculate the termination efficiency of the terminators.

Experimental Group:

(Inputs are measured by RFP expression and outputs are measured by GFP expression.)

Control Group:

(calibrates RFP input to GFP output.)

6. Experimental procedure

6.1 Media:

Lysogeny Broth (LB) liquid and solid media.

6.2 Protocol:

a. Plasmid pSB1A3 is chosen to be the vector that ligate GPF and RFP fragments. To protect the structural integrity of the constructed plasmid, we need to mutate a restriction enzyme cutting site named Pst I to Afl II using PCR. Proper primer are designed for this purpose.

PtoA-F:5'-CCACCTGACGTCTAAGAAAC-3'

PtoA-R:5'-ATGATCATCGCCGGCGAATTCAGGC-3'

b. Check whether the mutation of restriction cutting site is successful.

c.Transform the plasmid to competent cell DH5αand incubate overnight.

d. Pick up colonies, plasmid isolation, and digest to choose the positive one.

e. Amplify GFP & RFP. Do an electrophoresis for verification the PCR is successful.

f. Use specific restriction enzymes to digest plasmid mutant-psb1a3,GFP and RFP to get sticky ends and purify the DNA fragment after the Electrophoresis.

Digestion of plasmid mutant-pSB1A3 with Afl II and Not I;

Digestion of PCR product GFP with Not I and Spe I;

Digestion of PCR product RFP with Afl II and Spe I;

Put the tubes in 37℃ environment for 4-8 hours.

g. Ligation is needed to connect these 3 fragments together.

h.Transform the plasmid to competent cell DH5αand incubate overnight.

i.Pick up colonies, plasmid isolation, and choose the positive one.

j. Use restriction enzymes Xba I and Pst I to digest the constructed vector.

k. Ligation is needed to terminators to be measured and the vector together.

Transform the plasmid to competent cell and incubate overnight.

l. Transform the plasmid to competent cell DH5αand incubate overnight.

m. Test the fluorescent using the method of flow cytometry.

7. Parts used to create the terminator characterization.

8. Calculate terminator efficiency

TE=1-GE/GC

TE: Terminator efficiency

GE: Mean value of GFP from experimental group

GC: Mean value of GFP from control group

Under the ideal circumstances, the output of GFP and RFP should be almost the same and the value of TE should approach 0. If a strong terminator is placed into characterization experimental group plasmid, the only output should be RFP and the value of TE should approach 1.

9. Author’s contact information

Jiankui He: jiankuihe@gmail.com

Shui Jing: betty.jingshui@gmail.com

Mengshi Zhang: mengshi0928@gmail.com

Xianggeng Liu: liuxianggeng@gmail.com

Yao Chen: chen.yy@live.com

Boyu Qie: boyuqie@gmail.com

Liuxing Shen: shenliuxing@gmail.com

Zishan Wu: zishan.woo@gmail.com

10. References

[1] http://partsregistry.org/wiki/index.php/Part_Types:Measurement_Systems

[2] Haiyao Huang “Design and Characterization of Artificial Transcriptional Terminators”.

[3] Lars Velten, Nao” Units for Promoter Measurement in Mammalian Cells”