IGEM:IMPERIAL/2008/Prototype/Wetlab/test constructs: Difference between revisions

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__NOTOC__
===''Phase 1''===
===<center>''Phase 1''</center>===
<font size=2><font color=#001199>'''''Characterisation of Constitutive promoter and RBS'''''</font>
{| border="0" style="background:transparent;"  
{| border="0" style="background:transparent;"  
|Varying combinations of constitutive promoters and RBS will be characterized to identify an efficient  combination giving high levels of protein expression. In total Four combinations will be tried(Promoter-RBS),
|Width="400px"|<font size=2><font color=#0088DD>'''''1.1 Characterisation of Constitutive promoter and RBS'''''</font> <br><br>'''Aim :''' To characterise the different combinations of constitutive promoters and RBS in terms of steady-state protein production. At the end of this testing we will have decided upon a constitutive promoter and RBS combination to use in later test constructs. The combinations of Promoter and RBS are listed below,
#p43-gsiB,
#p43-gsiB,
#p43-SpoVG,  
#p43-SpoVG,  
#pVeg-gsiB,
#pVeg-gsiB,
#pVeg-SpoVG
#pVeg-SpoVG
|
|width="50px"|
|[[Image:Phase 1.PNG|thumb|300px]]
|[[Image:Phase 1.PNG|thumb|300px]]
|}
|}


===''Phase 2''===
===<center><font size=4.5>''Phase 2''</font></center>===
'''''Characterisation of inducible promoters and RBS'''''
 
{| border="0" style="background:transparent;"
{| border="0" style="background:transparent;"
|Varying combinations of inducible promoters and RBS will be characterized to identify an efficient  combination giving high levels of protein expression. In addition, constitutive promoters previously characterised will be used to express the transcriptional regulators required for each promoter. In total Four combinations will be tried(Promoter-RBS),
|Width="400px"|<font size=2><font color=#0088DD>'''''2.1 Characterisation of inducible promoters and RBS''''' </font><br><br>'''Aim:''' To characterise the different combinations of inducible promoters and RBS in terms of response time, transfer function and maximum steady-state level of protein. Each of these constructs required the use of the constitutive promoter-RBS to express the transcriptional regulators. By the end of this testing, our extensive characterisation of the different combinations would allow us to make a decision on which inducible promoter to use for Phase 3.
#pHyper-spank-gsiB,
#pHyper-spank-gsiB,
#pHyper-spank-SpoVG,  
#pHyper-spank-SpoVG,  
#pxyl-gsiB,
#pxyl-gsiB,
#pxyl-SpoVG
#pxyl-SpoVG
|
|width="50px"|
|[[Image:Phase 2-linduced.PNG|thumb|300px]]
|[[Image:Phase 2-linduced.PNG|thumb|300px]]
|}
|}


'''''Characterisation of light inducible promoters and RBS'''''  
{| border="0" style="background:transparent;" </font>
{| border="0" style="background:transparent;"
|Width="400px"|<font size=2><font color=#0088DD>'''''2.2 Characterisation of light inducible promoters and RBS''''' </font><br><br>'''Aim:''' Varying combinations of light inducible promoters and RBS will be characterized to identify an efficient  combination giving high levels of protein expression. In addition, constitutive promoters previously characterised will be used to express the light receptor protein YtvA required for efficient light sensing. In total, 4 combinations will be tried (Promoter-RBS),
|Varying combinations of light inducible promoters and RBS will be characterized to identify an efficient  combination giving high levels of protein expression. In addition, constitutive promoters previously characterised will be used to express the light receptor protein YtvA required for efficient light sensing. In total Four combinations will be tried(Promoter-RBS),
#pctc-gsiB,
#pctc-gsiB,
#pctc-SpoVG,  
#pctc-SpoVG,  
#pgsiB-gsiB,
#pgsiB-gsiB,
#pgsiB-SpoVG
#pgsiB-SpoVG
|
|width="50px"|
|[[Image:Phase 2-light induced.PNG|thumb|300px]]
|[[Image:Phase 2-light induced.PNG|thumb|300px]]
|}
|}


===''Phase 3''===
===<center><font size=4.5>''Phase 3''</font></center>===
'''''Characterisation of the clutch'''''
{| border="0" style="background:transparent;"
{| border="0" style="background:transparent;"
|valign="top"|To characterise the clutch EpsE affect on motility, EpsE is placed downstream of an inducible promoter.
|Width="400px"|<font size=2><font color=#0088DD>'''''3.1 Characterisation of the clutch'''''</font> <br><br>To characterise the effect of expression of the clutch, EpsE, on motility, EpsE is placed downstream of an inducible promoter.
|width="50px"|
|[[Image:Phase 2-linduced22.PNG|thumb|300px|]]
|[[Image:Phase 2-linduced22.PNG|thumb|300px|]]
|}
|}
'''''Characterisation of Biomaterials'''''
<font size=2><font color=#0088DD>'''''3.2 Characterisation of Biomaterials'''''</font>
{| border="0" style="background:transparent;"
{| border="0" style="background:transparent;"
|valign="top"|Combinations of signal peptides (SP) and Biomaterials will be placed downstream of an inducible promoter to characterise the synthesis and secretion in ''B.subtilis''. The following combinations were used (SP-Biomaterial):
|Width="400px"|Combinations of signal peptides (SP) and Biomaterials will be placed downstream of an inducible promoter to characterise the synthesis and secretion in ''B.subtilis''. The following combinations were used (SP-Biomaterial):
#SacB-Elastin
#SacB-Elastin
#SacB-Elastin
#LipA-Elastin
#SacB-EAK16-11
#LipA-EAK16-11
#LipA-EAK16-11
#LipA-EAK16-11
|width="50px"|
|
|[[Image:Phase 2-biomaterials.PNG|thumb|300px]]
|[[Image:Phase 2-biomaterials.PNG|thumb|300px]]
|}
|}
<br>
<br>


===''Phase 4''===
===<center><font size=4.5>''Phase 4''</font></center>===
'''''Light induced Expression of the clutch'''''
{| border="0" style="background:transparent;"
{| border="0" style="background:transparent;"
|valign="top"|Aim: To test the blue light induced expression of the clutch, EpsE and the affct of this on motility.   
|Width="400px"|<font size=2><font color=#0088DD>'''''4.1 Light induced Expression of the clutch''''' </font> <br><br>Aim: To test the blue light induced expression of the clutch, EpsE and the affct of this on motility.   
|width="50px"|
|[[Image:Phase 4-clutch.PNG|thumb|300px|]]
|[[Image:Phase 4-clutch.PNG|thumb|300px|]]
|}
|}


{| border="0" style="background:transparent;"
|Width="400px"|<font size=2><font color=#0088DD>'''''4.2 Light induced Expression of Biomaterials'''''</font> <br><br>Aim: To test the blue light induced expression and secretion of Biomaterial.
|width="50px"|
|[[Image:Phase 4-clutch.PNG|thumb|300px|]]
|}






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Latest revision as of 06:06, 3 September 2008

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<html><a href=http://openwetware.org/wiki/IGEM:IMPERIAL/2008/Prototype><img width=50px src=http://openwetware.org/images/f/f2/Imperial_2008_Logo.png></img</a></html> Home The Project B.subtilis Chassis Wet Lab Dry Lab Notebook

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Phase 1

1.1 Characterisation of Constitutive promoter and RBS

Aim : To characterise the different combinations of constitutive promoters and RBS in terms of steady-state protein production. At the end of this testing we will have decided upon a constitutive promoter and RBS combination to use in later test constructs. The combinations of Promoter and RBS are listed below,
  1. p43-gsiB,
  2. p43-SpoVG,
  3. pVeg-gsiB,
  4. pVeg-SpoVG

Phase 2

2.1 Characterisation of inducible promoters and RBS

Aim: To characterise the different combinations of inducible promoters and RBS in terms of response time, transfer function and maximum steady-state level of protein. Each of these constructs required the use of the constitutive promoter-RBS to express the transcriptional regulators. By the end of this testing, our extensive characterisation of the different combinations would allow us to make a decision on which inducible promoter to use for Phase 3.
  1. pHyper-spank-gsiB,
  2. pHyper-spank-SpoVG,
  3. pxyl-gsiB,
  4. pxyl-SpoVG
2.2 Characterisation of light inducible promoters and RBS

Aim: Varying combinations of light inducible promoters and RBS will be characterized to identify an efficient combination giving high levels of protein expression. In addition, constitutive promoters previously characterised will be used to express the light receptor protein YtvA required for efficient light sensing. In total, 4 combinations will be tried (Promoter-RBS),
  1. pctc-gsiB,
  2. pctc-SpoVG,
  3. pgsiB-gsiB,
  4. pgsiB-SpoVG

Phase 3

3.1 Characterisation of the clutch

To characterise the effect of expression of the clutch, EpsE, on motility, EpsE is placed downstream of an inducible promoter.

3.2 Characterisation of Biomaterials

Combinations of signal peptides (SP) and Biomaterials will be placed downstream of an inducible promoter to characterise the synthesis and secretion in B.subtilis. The following combinations were used (SP-Biomaterial):
  1. SacB-Elastin
  2. LipA-Elastin
  3. SacB-EAK16-11
  4. LipA-EAK16-11


Phase 4

4.1 Light induced Expression of the clutch

Aim: To test the blue light induced expression of the clutch, EpsE and the affct of this on motility.
4.2 Light induced Expression of Biomaterials

Aim: To test the blue light induced expression and secretion of Biomaterial.



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