Team:MIT gateway

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  <li><a href="https://2010.igem.org/Team:MIT_bact">Bacterial Protocol</a><br></li>
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<div style="width:250px; margin: 10px; position: relative; top: -4px; left:-11px; display: block; float:right; padding: 7px; background-color: white;">
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    <ul><li><a href="https://2010.igem.org/Team:MIT_bconst">Biobrick Construction</a><br></li>
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<dl id="nav">
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        <li><a href="https://2010.igem.org/Team:MIT_bexp">Bacterial Experiments</a></li></ul>
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<dt><b>Bacterial Protocol</b></dt>
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  <li><a href="https://2010.igem.org/Team:MIT_mmethods">Mammalian Protocol</a></li>
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<dd>
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    <ul><li><a href="https://2010.igem.org/Team:MIT_mmethods">Microfluidics</a></li>
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<ul>
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        <li><a href="https://2010.igem.org/Team:MIT_mge">Genetic Engineering</a></li>
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<li><a href="https://2010.igem.org/Team:MIT_bconst">Biobrick Construction</a></li>
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        <li class="sel"><a href="https://2010.igem.org/Team:MIT_gateway">Gateway Cloning</a></li>
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<li><a href="https://2010.igem.org/Team:MIT_bexp">Bacterial Experiments</a></li>
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    </ul>
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</ul>
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</ul>
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</dd>
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<br>
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<dt><b>Mammalian Protocol</b></dt>
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<dd>
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<ul>
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<li><a href="https://2010.igem.org/Team:MIT_mmethods">Microfluidics</a></li>
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<li><a href="https://2010.igem.org/Team:MIT_mge">Genetic Engineering</a></li>
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<li><a href="https://2010.igem.org/Team:MIT_gateway">Gateway Cloning</a></li>
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</ul>
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</dd>
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<dt><b>Phage Protocol</b></dt>
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<dd>
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<ul>
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<li><a href="https://2010.igem.org/Team:MIT_phageprot">Basic Protocol</a></li>
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</ul>
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</dd>
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<table width=70%><tr><td><div class="bodybaby">bacterial biobrick construction</div></td>
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<table width=650px style="background-color: white; margin-top:5px; padding: 10px;">
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<tr><td><div class="bodybaby">Gateway Cloning</div></td>
<tr><td><br>The Mammalian team used Gateway cloning to assemble its composite parts.<br><br>
<tr><td><br>The Mammalian team used Gateway cloning to assemble its composite parts.<br><br>
<div class="outline">
<div class="outline">
-
  <a href="#general">1 General Gateway Protocol</a><br>
+
  <a href="#gateway">1 Gateway Cloning</a><br>
-
  <a href="#miniprep">2 Commented Protocol</a><br>
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  <a href="#commented">2 Commented Protocol</a><br>
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  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#virtual">2.1 Design PCR-Primers with attB1.1 and attB2.1 sites</a><br>
+
  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#design">2.1 Design PCR-Primers</a><br>
-
  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#digest">2.2 Gel Purify PCR Product</a><br>
+
  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#gel">2.2 Gel Purify PCR Product</a><br>
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  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#antarctic">2.3 Confirm PCR Product with attB1/2.1 and DONR(TM)clone</a><br>
+
  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#confirm">2.3 Confirm PCR Product</a><br>
-
  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#gel">2.4 Measure DNA</a><br>
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  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#measure">2.4 Measure DNA</a><br>
-
  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#extract">2.5 Calculate PCR Product Needed</a><br>
+
  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#calculate">2.5 Calculate PCR Product Needed</a><br>
-
  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#transform">2.6 Calculate volume of DONRTM  needed</a><br>
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  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#volume">2.6 Calculate volume of DONRTM  needed</a><br>
-
  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#gel">2.7 Prepare Gateway Reaction</a><br>
+
  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#prepare">2.7 Prepare Gateway Reaction</a><br>
-
  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#gel">2.8 Retrieve 5x BP Clonase II</a><br>
+
  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#retrieve">2.8 Retrieve 5x BP Clonase II</a><br>
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  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#gel">2.9 Add BP-Clonase to Gateway Reaction</a><br>
+
  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#add">2.9 Add BP-Clonase to Gateway Reaction</a><br>
-
  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#gel">2.10 Incubate</a><br>
+
  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#incubate">2.10 Incubate</a><br>
-
  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#gel">2.11 Add ProteinaseK</a><br>
+
  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#proteinasek">2.11 Add ProteinaseK</a><br>
-
  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#gel">2.12 Transform Bacteria</a><br>
+
  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#transform">2.12 Transform Bacteria</a><br>
-
  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#gel">2.13 Plate Bacteria</a><br>
+
  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#plate">2.13 Plate Bacteria</a><br>
-
  <a href="#miniprep">3 Known Issues</a><br>
+
  <a href="#known">3 Known Issues</a><br>
  <a href="#cite">4 Citation</a><br>
  <a href="#cite">4 Citation</a><br>
</div></td><tr><td><br>
</div></td><tr><td><br>
-
<div class="bodybaby" id="general">General Biobrick Construction Protocol</div><br>
+
<div class="bodybaby" id="gateway">Gateway Cloning</div><br>
 +
 
 +
<div class="bodybaby" id="commented">Commented Protocol</div><br>
 +
<b class="bolded" id="miniprep">Design PCR-Primers</b><br>
 +
The Gateway®  clones have a reading frame which should be kept. Design primers that the PCR product starts with a ATG and ends with a STOP-codon or the last aminoacid (if you want to make a fusion protein). Primer3plus  is a powerful tool helping you to pick primers with the right annealing temperature which should be 60°C. Try to avoid self similarity and other things as usual, but because you are very limited in the position of the primers (its start and stop), I only care about annealing temperature and give it a try. Then just add to the primer which binds the start codon the attB1.1-sequence at his 5' End . To the primer which binds the stop codon or the last aminoacid add the attB2.1-sequence at his 5' End . The open reading frame is indicated and you should change the last two NN to code for an aminoacid of your choice. Good luck for the PCR! Because of the long 5' overhang and the restrictions on picking the primers, getting the PCR to work can be tricky.<br><br>
 +
 
 +
<u>Improved and more efficient att sites used to recombine into pDONR 221:<br></u>
 +
attB1.1  GGG-GCA-ACT-TTg-tac-aaa-aaa-gtt-gNN<br>
 +
attB2.1  GG-GGC-AAC-TTT-GTA-CAA-Caa-agt-tgN<br><br>
 +
 
 +
<u>The original att sites used to recombine into pDONR 221:<br></u>
 +
attB1  GGGG-ACA-AGT-TTg-tac-aaa-aaa-gca-ggc-tNN<br>
 +
attB2  GGG-GAC-CAC-TTT-GTA-CAA-Gaa-agc-tgg-gtN<br><br>
 +
 
 +
<u>The att sites used to recombine into pDONR P4-P1R:<br></u>
 +
attB4  GGGG-ACA-ACT-TTg-tat-aga-aaa-gtt-gNN<br>
 +
attB1  GGG-GAC-TGC-TTT-TTT-GTA-Caa-act-tgN<br><br>
 +
 
 +
<u>The att sites used to recombine into pDONR P2R-P3:<br></u>
 +
attB2  GGGG-ACA-GCT-TTc-ttg-tac-aaa-gtg-gNN<br>
 +
attB3  GGG-GAC-AAC-TTT-GTA-TAA-Taa-agt-tgN <br><br>
 +
 
 +
<b class="bolded" id="gel">Gel Purify PCR Product</b><br>
 +
Purification of the PCR-product is needed to get rid of smaller side-products, which remove primer-dimers which can result in false positive colonies. Remember that you want to clone DNA, so the cutting should be made on the weakest UV-light available and as fast as possible. And of course you NEVER make a picture of the gel before. Use the kid for gel-purification available in your lab. <br><br>
 +
 
 +
<b class="bolded" id="confirm">Confirm PCR Product</b><br>
 +
You need a PCR product with the  attB1.1 and attB2.2 and the DONRTM vector MUST have attP1 and attP2 sites, or it will not work.<br><br>
 +
 
 +
The amount of plasmids is not soo important as in a multiple Gateway® reaction, because it is more efficient. If you want to optimize you can calculate equimolar amounts of both plasmids as described in the How to measure DNA. Here we use double the amount of DEST-vector, because most of the ones we use are round and about double the size of the ENTRTM clones. <br><br>
 +
 
 +
<b class="bolded" id="measure">Measure DNA</b><br>
 +
The amount of ENTRTM is not so important as in a multiple Gateway® reaction, because it is more efficient. If you want to optimize you can calculate equimolar amounts of both plasmids as described in the multiple Gateway® protocol. Here we use double the amount of DEST-vector, because most of the ones we use are round and about double the size of the ENTRTM clones. <br><br>
 +
 
 +
<b class="bolded" id="calculate">Calculate PCR Product Needed</b><br>
 +
ng needed = (length of the PCR product in bp) x 0.0165  <br><br>
 +
 
 +
<b class="bolded" id="volume">Calculate volume of DONTR(TM) needed</b><br>
 +
µl needed = 75 ng needed / (concentration in ng/µl)<br><br>
 +
 
 +
The DONR-vector should be tested for low background colonies (due to a mutated ccdB-gene) when transferred in DH5alpha-bacteria. <br><br>
 +
 
 +
<b class="bolded" id="prepare">Prepare Gateway Reaction</b><br>
 +
PCR-product&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;( ? ng)<br>
 +
pDONRTM-vector&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(75 ng)<br>
 +
add water to a total volume of<b> 4µl </b><br><br>
-
<b class="bolded" id="miniprep">Miniprep.</b><br>
+
<b class="bolded" id="retrieve">Retrieve 5x BP-Clonase II</b><br>
-
To extract DNA from cells, we used the QIAprep Spin Miniprep Kit according to protocol.<br><br>
+
...From -20&deg;C. It is most efficiently mixed by pipetting up and down, do not vortex. <br><br>
-
<b class="bolded" id="virtual">Virtual Restriction Mapping.</b><br>
+
<b class="bolded" id="add">Add BP-Clonase to Gateway Reaction</b><br>
-
The sequences of all our individual parts were entered into a shared Geneious library, and all of our Biobrick parts were created virtually parallel to actual construction. We used Geneious to find enzymes for restriction mapping and sequencing validation of parts.<br><br>
+
The enzymes looses 50% activity after 15 freeze-thaw cycles. The advantage of BP-ClonaseTMII is that it can be stored at -20 °C because it contains already the buffer. DO NOT LEAVE OUT.<br><br>
-
<b class="bolded" id="digest">DNA Digestion.</b><br>
+
<b class="bolded" id="incubate">Incubate</b><br>
-
For both restriction mapping and for digesting a plasmid to insert or extract a biobrick, the appropriate buffer was added 1:10 to a DNA sample and 10 units of the appropriate enzyme(s) were used per 1ug of DNA. The reactions were allowed to run at the temperature optimal for the enzyme for 1 hour, the enzymes were denatured at 65&deg;C for 20 minutes, and then the reactions were held at 16&deg;C.<br><br>
+
Incubation over-night will enhance the reaction ca. 5-10 fold. This is especially important for PCR products over 5.000 bp. <br><br>
-
<b class="bolded" id="antarctic">Antarctic Phosphatase.</b><br>
+
<b class="bolded" id="proteinasek">Add ProteinaseK</b><br>
-
To prevent recircularization of digested plasmids, we used NEB's Antarctic Phosphatase according to protocol.<br><br>
+
This step will enhance the reaction ca. 100 fold!!!!. This is different to the LR-reactions which are only enhanced 2 fold by adding the proteinase K!!!!
-
<b class="bolded" id="gel">Gel Electrophoresis.</b><br>
+
<b class="bolded" id="transform">Transform Bacteria</b><br>
-
To view lengths of DNA, Orange G 6x from NEB was added 1:3 to 300ng miniprepped (and sometimes digested) DNA and run against 5uL of Hyperladder I from NEB in the lanes of 1% UltraPure agarose (Invitrogen) gels in TAE buffer at 120V for 45 minutes.<br><br>
+
For electro competent cells use 1-2 µl, for chemical competent all. <br><br>
-
<b class="bolded" id="extract">DNA Gel Extraction.</b><br>
+
<b class="bolded" id="plate">Plate Bacteria</b><br>
-
To extract the DNA from the agarose gel, the QIAquick Gel Extraction kit was used according to protocol.<br><br>
+
The resulting ENTRTM-vectors are kanamycin resistant. <br>
-
<b class="bolded" id="transform">Transformation.</b><br>
+
<br><br><div class="bodybaby" id="known">Known Issues</div><br>
-
Aliquots of competent JM2.300 E.coli cells were thawed on ice for 8 minutes. Approximately 10ng of each plasmid were added to the competent cells and the tube was tapped to mix. The cells incubated on ice for 30 minutes after which they were heat shocked at 42&deg;C for exactly 30 seconds and then incubated on ice for 2 minutes. Each transformation received 0.9 mL of room temperature Super Optimal broth with Catabolite repression, and were shaken at 37&deg;C at 280 rpm for 60 minutes. On a LB plate with antibiotics that select for cells transformed with our DNA, 20 uL of the mixture was plated on one half, the rest of the cells were pelleted and plated on the other half. Plates incubated upright for 20 minutes then agar up for 12-16 hours at 37&deg;C.
+
<ul><li>  The reaction is very efficient. You can obtain about 200 colonies of which about 95 % are correct.</li>
 +
<li> BP reactions work better with linear templates like PCR-products. If you want to use plasmids, linearize them first with a suitable restriction enzyme.</li>
 +
<li> The obtained plasmids are big. To check for correct clones digest with Sty I and in parallel with Eco RI and Hind III. Compare the pattern of bands with the predicted band size to find the correct clones.</li></ul><br>
<div class="bodybaby" id="cite">Citation</div><br>
<div class="bodybaby" id="cite">Citation</div><br>

Latest revision as of 16:54, 27 October 2010

Gateway Cloning

The Mammalian team used Gateway cloning to assemble its composite parts.

1 Gateway Cloning
2 Commented Protocol
    2.1 Design PCR-Primers
    2.2 Gel Purify PCR Product
    2.3 Confirm PCR Product
    2.4 Measure DNA
    2.5 Calculate PCR Product Needed
    2.6 Calculate volume of DONRTM needed
    2.7 Prepare Gateway Reaction
    2.8 Retrieve 5x BP Clonase II
    2.9 Add BP-Clonase to Gateway Reaction
    2.10 Incubate
    2.11 Add ProteinaseK
    2.12 Transform Bacteria
    2.13 Plate Bacteria
3 Known Issues
4 Citation

Gateway Cloning

Commented Protocol

Design PCR-Primers
The Gateway® clones have a reading frame which should be kept. Design primers that the PCR product starts with a ATG and ends with a STOP-codon or the last aminoacid (if you want to make a fusion protein). Primer3plus is a powerful tool helping you to pick primers with the right annealing temperature which should be 60°C. Try to avoid self similarity and other things as usual, but because you are very limited in the position of the primers (its start and stop), I only care about annealing temperature and give it a try. Then just add to the primer which binds the start codon the attB1.1-sequence at his 5' End . To the primer which binds the stop codon or the last aminoacid add the attB2.1-sequence at his 5' End . The open reading frame is indicated and you should change the last two NN to code for an aminoacid of your choice. Good luck for the PCR! Because of the long 5' overhang and the restrictions on picking the primers, getting the PCR to work can be tricky.

Improved and more efficient att sites used to recombine into pDONR 221:
 attB1.1 GGG-GCA-ACT-TTg-tac-aaa-aaa-gtt-gNN
attB2.1 GG-GGC-AAC-TTT-GTA-CAA-Caa-agt-tgN

The original att sites used to recombine into pDONR 221:
 attB1 GGGG-ACA-AGT-TTg-tac-aaa-aaa-gca-ggc-tNN
attB2 GGG-GAC-CAC-TTT-GTA-CAA-Gaa-agc-tgg-gtN

The att sites used to recombine into pDONR P4-P1R:
 attB4 GGGG-ACA-ACT-TTg-tat-aga-aaa-gtt-gNN
attB1 GGG-GAC-TGC-TTT-TTT-GTA-Caa-act-tgN

The att sites used to recombine into pDONR P2R-P3:
 attB2 GGGG-ACA-GCT-TTc-ttg-tac-aaa-gtg-gNN
attB3 GGG-GAC-AAC-TTT-GTA-TAA-Taa-agt-tgN

Gel Purify PCR Product
Purification of the PCR-product is needed to get rid of smaller side-products, which remove primer-dimers which can result in false positive colonies. Remember that you want to clone DNA, so the cutting should be made on the weakest UV-light available and as fast as possible. And of course you NEVER make a picture of the gel before. Use the kid for gel-purification available in your lab.

Confirm PCR Product
You need a PCR product with the attB1.1 and attB2.2 and the DONRTM vector MUST have attP1 and attP2 sites, or it will not work.

The amount of plasmids is not soo important as in a multiple Gateway® reaction, because it is more efficient. If you want to optimize you can calculate equimolar amounts of both plasmids as described in the How to measure DNA. Here we use double the amount of DEST-vector, because most of the ones we use are round and about double the size of the ENTRTM clones.

Measure DNA
The amount of ENTRTM is not so important as in a multiple Gateway® reaction, because it is more efficient. If you want to optimize you can calculate equimolar amounts of both plasmids as described in the multiple Gateway® protocol. Here we use double the amount of DEST-vector, because most of the ones we use are round and about double the size of the ENTRTM clones.

Calculate PCR Product Needed
ng needed = (length of the PCR product in bp) x 0.0165

Calculate volume of DONTR(TM) needed
µl needed = 75 ng needed / (concentration in ng/µl)

The DONR-vector should be tested for low background colonies (due to a mutated ccdB-gene) when transferred in DH5alpha-bacteria.

Prepare Gateway Reaction
PCR-product             ( ? ng)
pDONRTM-vector     (75 ng)
add water to a total volume of 4µl

Retrieve 5x BP-Clonase II
...From -20°C. It is most efficiently mixed by pipetting up and down, do not vortex.

Add BP-Clonase to Gateway Reaction
The enzymes looses 50% activity after 15 freeze-thaw cycles. The advantage of BP-ClonaseTMII is that it can be stored at -20 °C because it contains already the buffer. DO NOT LEAVE OUT.

Incubate
Incubation over-night will enhance the reaction ca. 5-10 fold. This is especially important for PCR products over 5.000 bp.

Add ProteinaseK
This step will enhance the reaction ca. 100 fold!!!!. This is different to the LR-reactions which are only enhanced 2 fold by adding the proteinase K!!!! Transform Bacteria
For electro competent cells use 1-2 µl, for chemical competent all.

Plate Bacteria
The resulting ENTRTM-vectors are kanamycin resistant.


Known Issues

  • The reaction is very efficient. You can obtain about 200 colonies of which about 95 % are correct.
  • BP reactions work better with linear templates like PCR-products. If you want to use plasmids, linearize them first with a suitable restriction enzyme.
  • The obtained plasmids are big. To check for correct clones digest with Sty I and in parallel with Eco RI and Hind III. Compare the pattern of bands with the predicted band size to find the correct clones.

Citation

1. Untergasser A. “Cloning – Gateway BP-Reaction II” Untergasser's Lab. Summer 2006. (include here the date when you accessed these page). .

Retrieved from "http://2010.igem.org/Team:MIT_gateway"