"
Team:NRP-UEA-Norwich/Project Mo-Flipper
From 2014.igem.org
(Difference between revisions)
Amiddlemiss (Talk | contribs) |
Amiddlemiss (Talk | contribs) |
||
| Line 17: | Line 17: | ||
<link href="//maxcdn.bootstrapcdn.com/font-awesome/4.1.0/css/font-awesome.min.css" rel="stylesheet"> | <link href="//maxcdn.bootstrapcdn.com/font-awesome/4.1.0/css/font-awesome.min.css" rel="stylesheet"> | ||
<style type="text/css"> | <style type="text/css"> | ||
| - | body{font-family:'Open Sans',sans-serif;font-size:13px}header{box-shadow:0 6px 6px rgba(0,0,0,.3)}header .container{height:80px}header .logo{padding:10px 0 10px 80px}header .igem{position:absolute;top:20px;right:-50px;z-index:100}header .igem img{width:40px;height:40px}header ul{float:right;list-style:none}header ul li{display:inline-block;white-space:nowrap}header ul li a{display:block;padding:0 15px;height:80px;line-height:80px;color:#000;text-decoration:none!important;transition:background-color .2s linear}header ul li a:active,header ul li a:hover,header ul li a:visited{background-color:#ecffeb;text-decoration:none!important;color:#000}header ul.dropdown-menu li{display:block}header ul.dropdown-menu li a{height:auto;padding:6px 20px;text-decoration:none}section{padding:50px 0}section h1{font-weight:300;font-size:48px;padding:0 0 50px}.dropdown-menu{margin:0}ul.menu li.dropdown:hover>ul.dropdown-menu{display:block}.bx-wrapper img{display:block;max-width:100%;min-width:970px;margin:0 auto}.welcome{padding:80px 0;background:url(https://static.igem.org/mediawiki/2014/0/06/Uea_header-bg.png)}.about{background:url(https://static.igem.org/mediawiki/2014/d/d7/Uea_body-bg.png)}.about h2{font-size:18px;font-weight:700}.about p.project{font-size:14px;padding:10px}.about p.project:last-of-type{padding-bottom:40px}.about .row:first-of-type{margin-bottom:40px}.about .col-md-4 i{transition:color .2s linear}.about .col-md-4:hover i{color:#2ECC71}.team{background:url(https://static.igem.org/mediawiki/2014/d/d7/Uea_body-bg.png);transition:all .3s linear;padding:0 0 40px}.team .img-responsive{border-radius:20em;-webkit-border-radius:20em;-moz-border-radius:20em;-webkit-filter:grayscale(100%);-moz-filter:grayscale(100%);-ms-filter:grayscale(100%);filter:grayscale(100%);filter:gray;transition:all .3s linear;max-width:200px;margin:0 auto}.team .col-md-4:hover .img-responsive{-webkit-filter:none;-moz-filter:none;-ms-filter:none;filter:none}.team .row:first-of-type{margin-bottom:40px}.team h2{font-size:18px;font-weight:700;color:#019875}.team h3{font-size:13px;font-weight:700;color:#019875}.team .col-md-3{padding:0 30px}.team .meetourteam{margin-bottom:60px}.social{background:url(https://static.igem.org/mediawiki/2014/d/d7/Uea_body-bg.png)}.sponsors .col-md-2{display:table;height:90px}.sponsors .middle{display:table-cell;vertical-align:middle}.sponsors .row:first-of-type{margin-bottom:40px}.general{padding-top:0;background:url(https://static.igem.org/mediawiki/2014/d/d7/Uea_body-bg.png)}.title{background:url(https://static.igem.org/mediawiki/2014/0/06/Uea_header-bg.png)}.title h1,.title h2{text-align:center}.title h1{margin-top:0;padding:40px 0;margin-bottom:40px}.title h2{font-size:18px;color:# | + | body{font-family:'Open Sans',sans-serif;font-size:13px}header{box-shadow:0 6px 6px rgba(0,0,0,.3)}header .container{height:80px}header .logo{padding:10px 0 10px 80px}header .igem{position:absolute;top:20px;right:-50px;z-index:100}header .igem img{width:40px;height:40px}header ul{float:right;list-style:none}header ul li{display:inline-block;white-space:nowrap}header ul li a{display:block;padding:0 15px;height:80px;line-height:80px;color:#000;text-decoration:none!important;transition:background-color .2s linear}header ul li a:active,header ul li a:hover,header ul li a:visited{background-color:#ecffeb;text-decoration:none!important;color:#000}header ul.dropdown-menu li{display:block}header ul.dropdown-menu li a{height:auto;padding:6px 20px;text-decoration:none}section{padding:50px 0}section h1{font-weight:300;font-size:48px;padding:0 0 50px}.dropdown-menu{margin:0}ul.menu li.dropdown:hover>ul.dropdown-menu{display:block}.bx-wrapper img{display:block;max-width:100%;min-width:970px;margin:0 auto}.welcome{padding:80px 0;background:url(https://static.igem.org/mediawiki/2014/0/06/Uea_header-bg.png)}.about{background:url(https://static.igem.org/mediawiki/2014/d/d7/Uea_body-bg.png)}.about h2{font-size:18px;font-weight:700}.about p.project{font-size:14px;padding:10px}.about p.project:last-of-type{padding-bottom:40px}.about .row:first-of-type{margin-bottom:40px}.about .col-md-4 i{transition:color .2s linear}.about .col-md-4:hover i{color:#2ECC71}.team{background:url(https://static.igem.org/mediawiki/2014/d/d7/Uea_body-bg.png);transition:all .3s linear;padding:0 0 40px}.team .img-responsive{border-radius:20em;-webkit-border-radius:20em;-moz-border-radius:20em;-webkit-filter:grayscale(100%);-moz-filter:grayscale(100%);-ms-filter:grayscale(100%);filter:grayscale(100%);filter:gray;transition:all .3s linear;max-width:200px;margin:0 auto}.team .col-md-4:hover .img-responsive{-webkit-filter:none;-moz-filter:none;-ms-filter:none;filter:none}.team .row:first-of-type{margin-bottom:40px}.team h2{font-size:18px;font-weight:700;color:#019875}.team h3{font-size:13px;font-weight:700;color:#019875}.team .col-md-3{padding:0 30px}.team .meetourteam{margin-bottom:60px}.social{background:url(https://static.igem.org/mediawiki/2014/d/d7/Uea_body-bg.png)}.sponsors .col-md-2{display:table;height:90px}.sponsors .middle{display:table-cell;vertical-align:middle}.sponsors .row:first-of-type{margin-bottom:40px}.general{padding-top:0;background:url(https://static.igem.org/mediawiki/2014/d/d7/Uea_body-bg.png)}.title{background:url(https://static.igem.org/mediawiki/2014/0/06/Uea_header-bg.png)}.title h1,.title h2{text-align:center}.title h1{margin-top:0;padding:40px 0;margin-bottom:40px}.title h2{font-size:18px;color:#019875;margin:0 0 50px;padding-bottom:40px}.general p{margin:0 0 40px}.fa-8x{font-size:8em;color:#019875}.row .boxed-headline{width:100%;height:200px;background-color:#90C695}.container .overview{padding-bottom:40px}footer{color:#444;background-color:#FFFFF;padding:50px 0}footer ul.sponsors{float:left;width:100%;padding:0;margin:20px 0 0;list-style-type:none}footer ul.sponsors li{display:inline;padding:0 20px}footer ul.sponsors li img{width:90px}#top-section{border:0;height:14px;z-index:100;top:0;position:fixed;width:975px;left:50%;margin-left:-487px;background-color:transparent}#content,#globalWrapper{width:100%;height:100%;border:0;background-color:transparent;margin:0;padding:0;font-size:100%}#catlinks,#contentSub,#footer-box,#p-logo,#search-controls,#sitesub,.firstHeading,.printfooter,.visualClear{display:none}h1,h2,h3,h4,h5,h6{border-bottom:none}#bodyContent h1,#bodyContent h2{margin-bottom:40px}.col-md-1,.col-md-10,.col-md-11,.col-md-12,.col-md-2,.col-md-3,.col-md-4,.col-md-5,.col-md-6,.col-md-7,.col-md-8,.col-md-9{float:left}.col-md-12{width:100%}.col-md-11{width:91.66666667%}.col-md-10{width:83.33333333%}.col-md-9{width:75%}.col-md-8{width:66.66666667%}.col-md-7{width:58.33333333%}.col-md-6{width:50%}.col-md-5{width:41.66666667%}.col-md-4{width:33.33333333%}.col-md-3{width:25%}.col-md-2{width:16.66666667%}.col-md-1{width:8.33333333%}.container{min-width:970px}ul.bxslider{margin:0 !important} |
</style> | </style> | ||
</head> | </head> | ||
| Line 106: | Line 106: | ||
<div class="container"> | <div class="container"> | ||
<center> | <center> | ||
| + | <h2><font color=#019875 /><b>Current iGEM standard</b></font></h2> | ||
<p>As part of the registry standard at iGEM all parts need to be in the same format, much like a computer file. If the files are incompatible (ie, different parts) then they will not work with the rest of the registry. The iGEM registry standard is the BioBrick cloning method; the method utilises 4 type IIs restriction endonucleases (RENs): EcoRI and XbaI are used in the prefix (the sequence of DNA before the region of interest) and SpaI and PstI are used in the suffix (the sequence of DNA after the region of interest. Systematic use of the 4 type IIs RENs allows new sections of DNA to be inserted into others.</p> | <p>As part of the registry standard at iGEM all parts need to be in the same format, much like a computer file. If the files are incompatible (ie, different parts) then they will not work with the rest of the registry. The iGEM registry standard is the BioBrick cloning method; the method utilises 4 type IIs restriction endonucleases (RENs): EcoRI and XbaI are used in the prefix (the sequence of DNA before the region of interest) and SpaI and PstI are used in the suffix (the sequence of DNA after the region of interest. Systematic use of the 4 type IIs RENs allows new sections of DNA to be inserted into others.</p> | ||
| + | <h2><font color=#019875 /><b>A new standard</b></font></h2> | ||
<p>For our project (Green Canary) we utilised the GoldenGate method of cloning, for more information on the GoldenGate method please see our GoldenGate page here(insert link to page). As previously described the parts submitted to the registry need to be in the same format (BioBrick), but, as all our wet lab work was carried out in GoldenGate, we had to convert our parts to BioBrick for submission. We developed the MoFlipper (Modular Cloning Flipper) which provides users with a oneway swap between GoldenGate and BioBrick. The standard pSB1C3 plasmid was improved and altered to include the GoldenGate REN recognition sites allowing for (near)scarless transfer from GoldenGate to BioBrick. The modified pSB1C3 contains two divergently orientated BsaI sites between the 4 BioBrick sites which will allow the GoldenGate fragments to be dropped into the (now) BioBrick vector in a one-pot, one-step, digestion-ligation GoldenGate cloning reaction. The improved pSB1C3 part now contains the GoldenGate recognition site as well as a RFP coding device that will allow for red white selection of the transformants.</p> | <p>For our project (Green Canary) we utilised the GoldenGate method of cloning, for more information on the GoldenGate method please see our GoldenGate page here(insert link to page). As previously described the parts submitted to the registry need to be in the same format (BioBrick), but, as all our wet lab work was carried out in GoldenGate, we had to convert our parts to BioBrick for submission. We developed the MoFlipper (Modular Cloning Flipper) which provides users with a oneway swap between GoldenGate and BioBrick. The standard pSB1C3 plasmid was improved and altered to include the GoldenGate REN recognition sites allowing for (near)scarless transfer from GoldenGate to BioBrick. The modified pSB1C3 contains two divergently orientated BsaI sites between the 4 BioBrick sites which will allow the GoldenGate fragments to be dropped into the (now) BioBrick vector in a one-pot, one-step, digestion-ligation GoldenGate cloning reaction. The improved pSB1C3 part now contains the GoldenGate recognition site as well as a RFP coding device that will allow for red white selection of the transformants.</p> | ||
<p>As discussed in our GoldenGate page the modules have specific 5’ and 3’ overhangs which correspond to their final position within the level 1 and then 2 constructs. We have developed 4 MoFlippers that will accept constructs designated promoters (GGAG —> AATG), coding sequences (AATG —> GCTT), terminators (GCTT —> GCGT) and a whole construct (GGAG —> GCGT) using the 4 base pair overhangs shown.</p> | <p>As discussed in our GoldenGate page the modules have specific 5’ and 3’ overhangs which correspond to their final position within the level 1 and then 2 constructs. We have developed 4 MoFlippers that will accept constructs designated promoters (GGAG —> AATG), coding sequences (AATG —> GCTT), terminators (GCTT —> GCGT) and a whole construct (GGAG —> GCGT) using the 4 base pair overhangs shown.</p> | ||
Revision as of 15:39, 17 October 2014
Golden Gate Modular Flipper - 'Mo Flipper'
The little plasmid that could!
Current iGEM standard
As part of the registry standard at iGEM all parts need to be in the same format, much like a computer file. If the files are incompatible (ie, different parts) then they will not work with the rest of the registry. The iGEM registry standard is the BioBrick cloning method; the method utilises 4 type IIs restriction endonucleases (RENs): EcoRI and XbaI are used in the prefix (the sequence of DNA before the region of interest) and SpaI and PstI are used in the suffix (the sequence of DNA after the region of interest. Systematic use of the 4 type IIs RENs allows new sections of DNA to be inserted into others.
A new standard
For our project (Green Canary) we utilised the GoldenGate method of cloning, for more information on the GoldenGate method please see our GoldenGate page here(insert link to page). As previously described the parts submitted to the registry need to be in the same format (BioBrick), but, as all our wet lab work was carried out in GoldenGate, we had to convert our parts to BioBrick for submission. We developed the MoFlipper (Modular Cloning Flipper) which provides users with a oneway swap between GoldenGate and BioBrick. The standard pSB1C3 plasmid was improved and altered to include the GoldenGate REN recognition sites allowing for (near)scarless transfer from GoldenGate to BioBrick. The modified pSB1C3 contains two divergently orientated BsaI sites between the 4 BioBrick sites which will allow the GoldenGate fragments to be dropped into the (now) BioBrick vector in a one-pot, one-step, digestion-ligation GoldenGate cloning reaction. The improved pSB1C3 part now contains the GoldenGate recognition site as well as a RFP coding device that will allow for red white selection of the transformants.
As discussed in our GoldenGate page the modules have specific 5’ and 3’ overhangs which correspond to their final position within the level 1 and then 2 constructs. We have developed 4 MoFlippers that will accept constructs designated promoters (GGAG —> AATG), coding sequences (AATG —> GCTT), terminators (GCTT —> GCGT) and a whole construct (GGAG —> GCGT) using the 4 base pair overhangs shown.
The image shown below gives an example of how the Flipper is designed to work in this way. The additional sequences, inserted between the RFP and the BioBrick prefix and suffix, are inverted BsaI sites as shown in orange. This enables the flipper to accept MoClo Pro + 5U parts, which begin GGAG and end AATG. The other 3 flippers work in much the same way but are designed with overhangs to accept coding sequence parts, terminator parts and whole transcriptional units as detailed above.
Figure 1: Promoter Mo flipper showing the inverted BsaI sites that allow the module to accept Pro + 5U parts, converting GoldenGate promoter parts into Biobrick form.
As GoldenGate uses type IIs restriction enzymes (that cleave outside of their recognition sequence), the sequence or part can be inserted into the MoFlipper, and not leave the recognition sites of BsaI, and then is fully functional as a BioBrick.
The NRP-UEA iGEM team are submitting this as an improved part of the pSB1C3 backbone used as the registry standard to allow more teams to work in the GoldenGate method of cloning whilst still being able to submit parts to the registry in the standard. The improved parts Bba_K1467100 (Promoter module), Bba_K1467200 (Coding sequence module), Bba_K1467300 (Terminator module), Bba_K1467400 (Transcription unit module) are detailed on our Parts Page.
