Team:Michigan/Project/Fabrication

From 2008.igem.org

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= '''<font color=dodgerblue size=6>Project Fabrication</font>''' =
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= '''<font color=royalblue size=6>Project Fabrication</font>''' =
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Prabhjyot's shit here...
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<font size=3>We will be using landing pads to insert the Sequestillator onto the chromosome of E. coli. Noisy behavior has proven detrimental to clock studies throughout the past, and we hope to reduce the noise in our system using landing pads.</font>
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<div align=center>[[Image:Phase1 small.png]]           [[Image:Phase2 small.png]]</div>
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 +
==<font size=5 color=royalblue>Landing Pad Plans for Sequestillator</font>==
 +
 
 +
 
 +
{|class="wikitable" border="0" cellpadding="10" cellspacing="1" style="padding: 1px; background-color:dodgerblue;  border: 1px solid mediumblue; text-align:center"
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!width="10%" align="justify" valign="top" style="background:gold; color:navy"|
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==<font color=royalblue size=3>Operon</font>==
 +
<br><br><font size=3>Activator Operon</font><br><br><br>
 +
 
 +
==  ==
 +
<br><br><font size=3>Repressor Operon</font>
 +
 
 +
!width="10%" align="justify" valign="top" style="background:gold; color:navy"|
 +
 
 +
==<font color=royalblue size=3>Topology</font>==
 +
 
 +
<div align=center>[[Image:New 1st operon - gold.PNG|220px]]<br><br>
 +
 
 +
==  ==
 +
<br>[[Image:New 2nd operon - gold.PNG|300px]]</div>
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 +
!width="10%" align="justify" valign="top" style="background:gold; color:navy"|
 +
 
 +
==<font color=royalblue size=3>Landing Pad</font>==
 +
 
 +
<div align=left><font size=3>Leucine Landing Pad</font></div><br>constructed by former Ninfa lab member, Don Eun Chang
 +
<br>
 +
 
 +
==  ==
 +
<div align=left><font size=3>Arabinose Landing Pad</font></div><br> iGEM 2007 project by Alyssa Delke & Khalid Miri
 +
 
 +
|}
 +
 
 +
 
 +
==<font size=5 color=royalblue>Specific Fabrication Techniques</font>==
 +
 
 +
 
 +
==<font size=4 color=royalblue>Activator Operon</font>==
 +
 
 +
{| class="wikitable" border="0" cellpadding="10" cellspacing="1" style="padding: 5px; background-color:transparent;  border: 1px solid transparent;text-align:center"
 +
!width="70%" align="justify" valign="top" style="background:transparent; color:navy"|
 +
 
 +
<br><font size=3 color=royalblue>Constitutive Promoter</font>
 +
*pArabLP was [https://2008.igem.org/Team:Michigan/Notebook/DigestProtocol digested] with EcoRI and NdeI
 +
*BioBrick constitutive promoters were double [https://2008.igem.org/Team:Michigan/Notebook/DigestProtocol digested] EcoRI and NdeI.
 +
*Digests were [https://2008.igem.org/Team:Michigan/Notebook/DigestProtocol purified] using QIAGEN Purification Kit
 +
*[https://2008.igem.org/Team:Michigan/Notebook/LigationProtocol Ligation] was done overnight in 4⁰C.
 +
*pArabLP-CP was then [https://2008.igem.org/Team:Michigan/Notebook/TransformationProtocol transformed] into E.coli competent cells and grown overnight on LB+Amp+Cm plates in 37⁰C.
 +
*Single colonies were isolated from the plates and grown in 5mL liquid culture containing LB+Amp+Cm overnight in 37⁰C.
 +
*Plasmids from cells were [https://2008.igem.org/Team:Michigan/Notebook/PlasmidPrepProtocol isolated] by using QIAGEN MiniPrep Kit
 +
*Plasmids were then [https://2008.igem.org/Team:Michigan/Notebook/DigestProtocol digested] with EcoRI and SpeI  overnight in 37⁰C (to ensure proper ligation).
 +
*[https://2008.igem.org/Team:Michigan/Notebook/GelProtocol Analytical gel] was then run to check fragment size of approximately 50bp.
 +
 
 +
<br><br><font size=3 color=royalblue>NifA</font>
 +
*NifA was [https://2008.igem.org/Team:Michigan/Notebook/PCRProtocol amplified] from pRT22 using primers that had EcoRI, XbaI, NdeI and SpeI, PstI restriction sites flanking the 1560bp mutant sequence.
 +
*The NifA sequence was then [https://2008.igem.org/Team:Michigan/Notebook/PCRProtocol purified] using the QIAGEN QIAquick PCR Purification Kit.
 +
*NifA was double [https://2008.igem.org/Team:Michigan/Notebook/DigestProtocol digested] with NdeI and SpeI and left overnight in 37⁰C.
 +
*pArabLP-CP was double [https://2008.igem.org/Team:Michigan/Notebook/DigestProtocol digested] with NdeI and SpeI also overnight at 37⁰C.
 +
*Digests were [https://2008.igem.org/Team:Michigan/Notebook/DigestProtocol purified] using QIAGEN Purification Kit
 +
*[https://2008.igem.org/Team:Michigan/Notebook/LigationProtocol Ligation] was done overnight in 4⁰C.
 +
*pArabLP-NifA was then [https://2008.igem.org/Team:Michigan/Notebook/TransformationProtocol transformed] into E.coli competent cells and grown overnight on LB+Amp+Cm plates in 37⁰C.
 +
*Single colonies were isolated from the plates and grown in 5mL liquid culture containing LB+Amp+Cm overnight in 37⁰C.
 +
*Plasmids from cells were [https://2008.igem.org/Team:Michigan/Notebook/PlasmidPrepProtocol isolated] by using QIAGEN MiniPrep Kit
 +
*Plasmids were then [https://2008.igem.org/Team:Michigan/Notebook/DigestProtocol digested] with EcoRI and SpeI  overnight in 37⁰C (to ensure proper ligation).
 +
*[https://2008.igem.org/Team:Michigan/Notebook/GelProtocol Analytical gel] was then run to check fragment size of approximately 1650bp.
 +
 
 +
 
 +
!width="10%" align="justify" valign="top" style="background:transparent; color:navy"|
 +
 
 +
<br><br><br>[[Image:Fabrication - constitutive promoter.PNG|400px]]
 +
<br><br><br><br><br><br><br><br><br>[[Image:Fabrication - nifA.PNG|400px]]
 +
 
 +
|}
 +
 
 +
==<font size=4 color=royalblue>Repressor Operon</font>==
 +
 
 +
{| class="wikitable" border="0" cellpadding="10" cellspacing="1" style="padding: 5px; background-color:transparent;  border: 1px solid transparent;text-align:center"
 +
!width="70%" align="justify" valign="top" style="background:transparent; color:navy"|
 +
 
 +
<font size=3 color=royalblue>NifHp</font>
 +
*The NifH promoter (NifHp) was [https://2008.igem.org/Team:Michigan/Notebook/PCRProtocol amplified] from pRT22 using primers that had EcoRI, XbaI and NdeI, SpeI, PstI restriction sites flanking the 290bp sequence.
 +
*The NifHp sequence was then [https://2008.igem.org/Team:Michigan/Notebook/PCRProtocol purified] using the QIAGEN QIAquick PCR Purification Kit.
 +
*NifHp was double [https://2008.igem.org/Team:Michigan/Notebook/DigestProtocol digested] with EcoRI and SpeI and left overnight in 37⁰C.
 +
*pLeuLP was double [https://2008.igem.org/Team:Michigan/Notebook/DigestProtocol digested] with EcoRI and SpeI also overnight at 37⁰C.
 +
*Digests were [https://2008.igem.org/Team:Michigan/Notebook/DigestProtocol purified] using QIAGEN Purification Kit
 +
*[https://2008.igem.org/Team:Michigan/Notebook/LigationProtocol Ligation] was done overnight in 4⁰C. Newly formed plasmid is renamed to pCLOCK1
 +
*pCLOCK1 was then [https://2008.igem.org/Team:Michigan/Notebook/TransformationProtocol  transformed] into E.coli competent cells and grown overnight on LB+Amp+Cm plates in 37⁰C.
 +
*Single colonies were isolated from the plates and grown in 5mL liquid culture containing LB+Amp+Cm overnight in 37⁰C.
 +
*Plasmids from cells were [https://2008.igem.org/Team:Michigan/Notebook/PlasmidPrepProtocol isolated] by using QIAGEN MiniPrep Kit
 +
*Plasmids were then [https://2008.igem.org/Team:Michigan/Notebook/DigestProtocol digested] with EcoRI and SpeI overnight in 37⁰C (to ensure proper ligation).
 +
*[https://2008.igem.org/Team:Michigan/Notebook/GelProtocol Analytical gel] was then run to check fragment size of approximately 300bp.
 +
 
 +
<br><br><font size=3 color=royalblue>NifL</font>
 +
*NifL was [https://2008.igem.org/Team:Michigan/Notebook/PCRProtocol amplified] from pIM18 using primers that had EcoRI, XbaI, NdeI and SpeI, PstI restriction sites flanking the 1590bp mutant sequence.
 +
*The NifL sequence was then [https://2008.igem.org/Team:Michigan/Notebook/PCRProtocol purified] using the QIAGEN QIAquick PCR Purification Kit.
 +
*NifL was double [https://2008.igem.org/Team:Michigan/Notebook/DigestProtocol digested] with NdeI and SpeI and left overnight in 37⁰C.
 +
*pLeuLP with NifHP was double [https://2008.igem.org/Team:Michigan/Notebook/DigestProtocol digested] with NdeI and SpeI also overnight at 37⁰C.
 +
*Digests were [https://2008.igem.org/Team:Michigan/Notebook/DigestProtocol purified] using QIAGEN Purification Kit
 +
*[https://2008.igem.org/Team:Michigan/Notebook/LigationProtocol Ligation] was done overnight in 4⁰C. Newly formed plasmid is renamed to pCLOCK2
 +
*pCLOCK2 was then [https://2008.igem.org/Team:Michigan/Notebook/TransformationProtocol transformed] into E.coli competent cells and grown overnight on LB+Amp+Cm plates in 37⁰C.
 +
*Single colonies were isolated from the plates and grown in 5mL liquid culture containing LB+Amp+Cm overnight in 37⁰C.
 +
*Plasmids from cells were [https://2008.igem.org/Team:Michigan/Notebook/PlasmidPrepProtocol isolated] by using QIAGEN MiniPrep Kit
 +
*Plasmids were then [https://2008.igem.org/Team:Michigan/Notebook/DigestProtocol digested] with EcoRI and SpeI  overnight in 37⁰C (to ensure proper ligation).
 +
*[https://2008.igem.org/Team:Michigan/Notebook/GelProtocol Analytical gel] was then run to check fragment size of approximately 2000bp.
 +
 
 +
<br><br><font size=3 color=royalblue>GFP </font>
 +
*pClock2 was [https://2008.igem.org/Team:Michigan/Notebook/DigestProtocol digested] with EcoRI and SpeI
 +
*BioBrick BBa_E0040 that contained GFP was [https://2008.igem.org/Team:Michigan/Notebook/DigestProtocol digested] with EcoRI and XbaI in sequential digests.
 +
*Digests were [https://2008.igem.org/Team:Michigan/Notebook/DigestProtocol purified] using QIAGEN Purification Kit
 +
*[https://2008.igem.org/Team:Michigan/Notebook/LigationProtocol Ligation] into pLeuLP with NifHP and NifL was done overnight in 4⁰C.
 +
*pCLOCK2 was then [https://2008.igem.org/Team:Michigan/Notebook/TransformationProtocol transformed] into E.coli competent cells and grown overnight on LB+Amp plates in 37⁰C.
 +
*Single colonies were isolated from the plates and grown in 5mL liquid culture containing LB+Amp overnight in 37⁰C.
 +
*Plasmids from cells were [https://2008.igem.org/Team:Michigan/Notebook/PlasmidPrepProtocol isolated] by using QIAGEN MiniPrep Kit
 +
*Plasmids were then [https://2008.igem.org/Team:Michigan/Notebook/DigestProtocol digested] with EcoRI and SpeI  overnight in 37⁰C (to ensure proper ligation).
 +
*[https://2008.igem.org/Team:Michigan/Notebook/GelProtocol Analytical gel] was then run to check fragment size of approximately 2700bp.
 +
 
 +
!width="10%" align="justify" valign="top" style="background:transparent; color:navy"|
 +
 
 +
<br><br><br><br>[[Image:Fabrication - nifHP.PNG|400px]]
 +
<br><br><br><br><br><br><br><br><br><br><br><br><br>[[Image:Fabrication - nifL.PNG|400px]]
 +
<br><br><br><br><br><br><br><br><br><br><br><br>[[Image:Fabrication - GFP.PNG|400px]]
 +
|}
 +
 
 +
 
 +
 
 +
<div align=center>For better visualization of what our parts look like in our landing pads, see our [[Team:Michigan/Project/LandingPads | Landing Pad Page]]</div>
|}
|}

Latest revision as of 03:53, 30 October 2008


Michigan iGEM website header.jpg

HOME THE TEAM THE PROJECT REGISTRY PARTS NOTEBOOK


Project Fabrication

We will be using landing pads to insert the Sequestillator onto the chromosome of E. coli. Noisy behavior has proven detrimental to clock studies throughout the past, and we hope to reduce the noise in our system using landing pads.


Landing Pad Plans for Sequestillator

Operon



Activator Operon




Repressor Operon

Topology

New 1st operon - gold.PNG


New 2nd operon - gold.PNG

Landing Pad

Leucine Landing Pad

constructed by former Ninfa lab member, Don Eun Chang


Arabinose Landing Pad

iGEM 2007 project by Alyssa Delke & Khalid Miri


Specific Fabrication Techniques

Activator Operon


Constitutive Promoter

  • pArabLP was digested with EcoRI and NdeI
  • BioBrick constitutive promoters were double digested EcoRI and NdeI.
  • Digests were purified using QIAGEN Purification Kit
  • Ligation was done overnight in 4⁰C.
  • pArabLP-CP was then transformed into E.coli competent cells and grown overnight on LB+Amp+Cm plates in 37⁰C.
  • Single colonies were isolated from the plates and grown in 5mL liquid culture containing LB+Amp+Cm overnight in 37⁰C.
  • Plasmids from cells were isolated by using QIAGEN MiniPrep Kit
  • Plasmids were then digested with EcoRI and SpeI overnight in 37⁰C (to ensure proper ligation).
  • Analytical gel was then run to check fragment size of approximately 50bp.



NifA

  • NifA was amplified from pRT22 using primers that had EcoRI, XbaI, NdeI and SpeI, PstI restriction sites flanking the 1560bp mutant sequence.
  • The NifA sequence was then purified using the QIAGEN QIAquick PCR Purification Kit.
  • NifA was double digested with NdeI and SpeI and left overnight in 37⁰C.
  • pArabLP-CP was double digested with NdeI and SpeI also overnight at 37⁰C.
  • Digests were purified using QIAGEN Purification Kit
  • Ligation was done overnight in 4⁰C.
  • pArabLP-NifA was then transformed into E.coli competent cells and grown overnight on LB+Amp+Cm plates in 37⁰C.
  • Single colonies were isolated from the plates and grown in 5mL liquid culture containing LB+Amp+Cm overnight in 37⁰C.
  • Plasmids from cells were isolated by using QIAGEN MiniPrep Kit
  • Plasmids were then digested with EcoRI and SpeI overnight in 37⁰C (to ensure proper ligation).
  • Analytical gel was then run to check fragment size of approximately 1650bp.





Fabrication - constitutive promoter.PNG








Fabrication - nifA.PNG

Repressor Operon

NifHp

  • The NifH promoter (NifHp) was amplified from pRT22 using primers that had EcoRI, XbaI and NdeI, SpeI, PstI restriction sites flanking the 290bp sequence.
  • The NifHp sequence was then purified using the QIAGEN QIAquick PCR Purification Kit.
  • NifHp was double digested with EcoRI and SpeI and left overnight in 37⁰C.
  • pLeuLP was double digested with EcoRI and SpeI also overnight at 37⁰C.
  • Digests were purified using QIAGEN Purification Kit
  • Ligation was done overnight in 4⁰C. Newly formed plasmid is renamed to pCLOCK1
  • pCLOCK1 was then transformed into E.coli competent cells and grown overnight on LB+Amp+Cm plates in 37⁰C.
  • Single colonies were isolated from the plates and grown in 5mL liquid culture containing LB+Amp+Cm overnight in 37⁰C.
  • Plasmids from cells were isolated by using QIAGEN MiniPrep Kit
  • Plasmids were then digested with EcoRI and SpeI overnight in 37⁰C (to ensure proper ligation).
  • Analytical gel was then run to check fragment size of approximately 300bp.



NifL

  • NifL was amplified from pIM18 using primers that had EcoRI, XbaI, NdeI and SpeI, PstI restriction sites flanking the 1590bp mutant sequence.
  • The NifL sequence was then purified using the QIAGEN QIAquick PCR Purification Kit.
  • NifL was double digested with NdeI and SpeI and left overnight in 37⁰C.
  • pLeuLP with NifHP was double digested with NdeI and SpeI also overnight at 37⁰C.
  • Digests were purified using QIAGEN Purification Kit
  • Ligation was done overnight in 4⁰C. Newly formed plasmid is renamed to pCLOCK2
  • pCLOCK2 was then transformed into E.coli competent cells and grown overnight on LB+Amp+Cm plates in 37⁰C.
  • Single colonies were isolated from the plates and grown in 5mL liquid culture containing LB+Amp+Cm overnight in 37⁰C.
  • Plasmids from cells were isolated by using QIAGEN MiniPrep Kit
  • Plasmids were then digested with EcoRI and SpeI overnight in 37⁰C (to ensure proper ligation).
  • Analytical gel was then run to check fragment size of approximately 2000bp.



GFP

  • pClock2 was digested with EcoRI and SpeI
  • BioBrick BBa_E0040 that contained GFP was digested with EcoRI and XbaI in sequential digests.
  • Digests were purified using QIAGEN Purification Kit
  • Ligation into pLeuLP with NifHP and NifL was done overnight in 4⁰C.
  • pCLOCK2 was then transformed into E.coli competent cells and grown overnight on LB+Amp plates in 37⁰C.
  • Single colonies were isolated from the plates and grown in 5mL liquid culture containing LB+Amp overnight in 37⁰C.
  • Plasmids from cells were isolated by using QIAGEN MiniPrep Kit
  • Plasmids were then digested with EcoRI and SpeI overnight in 37⁰C (to ensure proper ligation).
  • Analytical gel was then run to check fragment size of approximately 2700bp.





Fabrication - nifHP.PNG












Fabrication - nifL.PNG











Fabrication - GFP.PNG


For better visualization of what our parts look like in our landing pads, see our Landing Pad Page