Difference between revisions of "PBAD SPL"
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Here the DTU iGEM team will present their work on characterizing a SPL of pBAD promoters.
+
=Description=
 +
This is a page containing a synthetic promoter library (SPL) of pBAD promoters made by [http://2013.igem.org/Team:DTU-Denmark 2013 DTU iGEM team]. The library can be used to make and arabinose inducible system with very low leakiness, or tune the the promoter strength of the system when it's turned on.
  
−
List of sequences:
+
A tight inducible system can be a very useful tool to express lethal or growth inhibiting proteins in ''E.coli''. It can also be necessary with a non leaky expression system if you would like to visualize the expression of GFP SF in ''E.coli'' (see [http://2013.igem.org/Team:DTU-Denmark/HelloWorld link]). 
 +
 
 +
 
 +
 
 +
=The library=
 +
Here is a complete list of the sequences and their relative leakiness and strength:
  
−
==DTU pBAD SPL==
 
 
{|class="sortable" style="border:3px solid black; width: 70%" align="center"  cellspacing="0"
 
{|class="sortable" style="border:3px solid black; width: 70%" align="center"  cellspacing="0"
 
! style="background:#AAAAAA; color:black"  |Identifier
 
! style="background:#AAAAAA; color:black"  |Identifier
 
! style="background:#AAAAAA; color:black"  |Sequence<sup>a</sup>
 
! style="background:#AAAAAA; color:black"  |Sequence<sup>a</sup>
−
! colspan="2" style="background:#AAAAAA; color:black"  |Induced Strength<sup>b</sup>
+
! style="background:#AAAAAA; color:black" |Induced Strength
−
! colspan="2" style="background:#AAAAAA; color:black"  |Leakyness<sup>b</sup>
+
! style="background:#AAAAAA; color:black"  |Leakiness
 +
! colspan="3" style="background:#AAAAAA; color:black"  |Inducibility<sup>b</sup>
 +
 
 
|-
 
|-
−
|align="center"|Col. 2
+
|align="center"|Col. 02
−
|<font face="Courier" size="+1">CTGACGACGCCCCTCTCCGCCCCTTAAAATGTCCA</font>
+
|<font face="Courier" size="+1" class=">CTGACGACGCCCCTCTCCGCCCCTTAAAATGTCCA</font>
−
|align="center"|14.2
+
 
|align="center"|14.2
 
|align="center"|14.2
 +
|align="center"|0.203
 +
|align="center"|69.9
 
|-
 
|-
−
|align="center"|Col. 3
+
|align="center"|Col. 03
 
|<font face="Courier" size="+1">CTGACGTAATCATACCGCCGAAAGTATTATCATTA</font>
 
|<font face="Courier" size="+1">CTGACGTAATCATACCGCCGAAAGTATTATCATTA</font>
 
|align="center"|16.6
 
|align="center"|16.6
−
|align="center"|14.2
+
|align="center"|0.426
 +
|align="center"|39
 
|-
 
|-
−
|align="center"|Col. 4
+
|align="center"|Col. 04
 
|<font face="Courier" size="+1">CTGACGCGACAACATTGCGTCCTATAAAATGCCGA</font>
 
|<font face="Courier" size="+1">CTGACGCGACAACATTGCGTCCTATAAAATGCCGA</font>
 
|align="center"|14.2
 
|align="center"|14.2
−
|align="center"|14.2
+
|align="center"|0.345
 +
|align="center"|41
 
|-
 
|-
−
|align="center"|<partinfo>J23102</partinfo>
+
|align="center"|Col. 05
−
|<font face="Courier" size="+1">ttgacagctagctcagtcctaggta<font color="red">c</font>t<font color="red">g</font>tgctagc</font>
+
|<font face="Courier" size="+1">CTGACGGCCCGCCCCCGATGCGCATAAAATACCAA</font>
−
|align="center"|0.86
+
|align="center"|17.7
 +
|align="center"|0.424
 +
|align="center"|41.6
 
|-
 
|-
−
|align="center"|<partinfo>J23103</partinfo>
+
|align="center"|Col. 12
−
|<font face="Courier" size="+1"><font color="red">c</font>tga<font color="red">t</font>agctagctcagtcctagg<font color="red">g</font>attatgctagc</font>
+
|<font face="Courier" size="+1">CTGACGCGTATCGCGAGCGGGCGTTATTATACGCA</font>
−
|align="center"|0.01
+
|align="center"|13.6
 +
|align="center"|0.414
 +
|align="center"|32.9
 
|-
 
|-
−
|align="center"|<partinfo>J23104</partinfo>
+
|align="center"|Col. 10
−
|<font face="Courier" size="+1">ttgacagctagctcagtcctaggtatt<font color="red">g</font>tgctagc</font>
+
|<font face="Courier" size="+1">CTGACGCCCCGAATCAGTAGTATTTATTATCTAGA</font>
−
|align="center"|0.72
+
|align="center"|18
 +
|align="center"|0.462
 +
|align="center"|38.8
 
|-
 
|-
−
|align="center"|<partinfo>J23105</partinfo>
+
|align="center"|Col. 09
−
|<font face="Courier" size="+1">tt<font color="red">t</font>ac<font color="red">g</font>gctagctcagtcctaggta<font color="red">c</font>tatgctagc</font>
+
|<font face="Courier" size="+1">CTGACGCCACCCCCCCCCCCGGCGTATAATTCCCA</font>
−
|align="center"|0.24
+
|align="center"|16.1
 +
|align="center"|0.530
 +
|align="center"|30.4
 
|-
 
|-
−
|align="center"|<partinfo>J23106</partinfo>
+
|align="center"|Col. 08
−
|<font face="Courier" size="+1">tt<font color="red">t</font>ac<font color="red">g</font>gctagctcagtcctaggtat<font color="red">ag</font>tgctagc</font>
+
|<font face="Courier" size="+1">CTGACGCCCTACCGCTCGCCCCCCTATTATACCCA</font>
−
|align="center"|0.47
+
|align="center"|13.4
 +
|align="center"|0.211
 +
|align="center"|63.4
 
|-
 
|-
−
|align="center"|<partinfo>J23107</partinfo>
+
|align="center"|Col. 13
−
|<font face="Courier" size="+1">tt<font color="red">t</font>ac<font color="red">g</font>gctagctcag<font color="red">c</font>cctaggtattatgctagc</font>
+
|<font face="Courier" size="+1">CTGACGCCCCCCCCGTCCACCCCCTAATATCCCGA</font>
−
|align="center"|0.36
+
|align="center"|10.9
 +
|align="center"|0.096
 +
|align="center"|113.6
 
|-
 
|-
−
|align="center"|<partinfo>J23108</partinfo>
+
|align="center"|Col. 15
−
|<font face="Courier" size="+1"><font color="red">c</font>tgacagctagctcagtcctaggtat<font color="red">a</font>atgctagc</font>
+
|<font face="Courier" size="+1">CTGACGTGCGCCTGCCGTCCAAAGTAATATCCTTA</font>
−
|align="center"|0.51
+
|align="center"|15.5
 +
|align="center"|0.235
 +
|align="center"|65.8
 
|-
 
|-
−
|align="center"|<partinfo>J23109</partinfo>
+
|align="center"|Col. 18
−
|<font face="Courier" size="+1">tt<font color="red">t</font>acagctagctcagtcctagg<font color="red">g</font>a<font color="red">ctg</font>tgctagc</font>
+
|<font face="Courier" size="+1">CTGACGCCCCGAATCAGTAGTATTTATTATCTAGA</font>
−
|align="center"|0.04
+
|align="center"|19.8
 +
|align="center"|0.524
 +
|align="center"|37.8
 
|-
 
|-
−
|align="center"|<partinfo>J23110</partinfo>
+
|align="center"|Col. 19
−
|<font face="Courier" size="+1">tt<font color="red">t</font>ac<font color="red">g</font>gctagctcagtcctaggta<font color="red">ca</font>atgctagc</font>
+
|<font face="Courier" size="+1">CTGACGCTCGCACCGGCGACCCGATAATATCCATA</font>
−
|align="center"|0.33
+
|align="center"|18.0
 +
|align="center"|0.315
 +
|align="center"|57
 
|-
 
|-
−
|align="center"|<partinfo>J23111</partinfo>
+
|align="center"|Col. 31
−
|<font face="Courier" size="+1">ttgac<font color="red">g</font>gctagctcagtcctaggtat<font color="red">ag</font>tgctagc</font>
+
|<font face="Courier" size="+1">CTGACGCCCCCCCTCCCCCGACCTAAAATATCCAG</font>
−
|align="center"|0.58
+
|align="center"|3.8
 +
|align="center"|0.161
 +
|align="center"|23.6
 
|-
 
|-
−
|align="center"|<partinfo>J23112</partinfo>
+
|align="center"|Col. 29
−
|<font face="Courier" size="+1"><font color="red">c</font>tga<font color="red">t</font>agctagctcagtcctagg<font color="red">g</font>attatgctagc</font>
+
|<font face="Courier" size="+1">CTGACGCCGCTGCACCCGTCCCCCTATTATAGCAA</font>
−
|align="center"|0.00
+
|align="center"|14.8
 +
|align="center"|0.301
 +
|align="center"|49.2
 
|-
 
|-
−
|align="center"|<partinfo>J23113</partinfo>
+
|align="center"|Col. 26
−
|<font face="Courier" size="+1"><font color="red">c</font>tga<font color="red">tg</font>gctagctcagtcctagg<font color="red">g</font>attatgctagc</font>
+
|<font face="Courier" size="+1">CTGACGCCGATCCTATCTCCCTATTAAAATTTTTA</font>
−
|align="center"|0.01
+
|align="center"|13.8
 +
|align="center"|0.292
 +
|align="center"|47.2
 
|-
 
|-
−
|align="center"|<partinfo>J23114</partinfo>
+
|align="center"|Col. 22
−
|<font face="Courier" size="+1">tt<font color="red">t</font>a<font color="red">tg</font>gctagctcagtcctaggta<font color="red">ca</font>atgctagc</font>
+
|<font face="Courier" size="+1">CTGACGCCCCGAATCAGTAGTATTTATTATCTAGA</font>
−
|align="center"|0.10
+
|align="center"|16.8
 +
|align="center"|0.466
 +
|align="center"|36.1
 
|-
 
|-
−
|align="center"|<partinfo>J23115</partinfo>
+
|align="center"|Col. 33
−
|<font face="Courier" size="+1">tt<font color="red">t</font>a<font color="red">t</font>agctagctcag<font color="red">c</font>cct<font color="red">t</font>ggta<font color="red">ca</font>atgctagc</font>
+
|<font face="Courier" size="+1">CTGACGCCAGCTTCCTACTTCAAATATAATCCGTA</font>
−
|align="center"|0.15
+
|align="center"|14.4
 +
|align="center"|1.01
 +
|align="center"|14.3
 
|-
 
|-
−
|align="center"|<partinfo>J23116</partinfo>
+
|align="center"|Col. 34
−
|<font face="Courier" size="+1">ttgacagctagctcagtcctagg<font color="red">g</font>a<font color="red">c</font>tatgctagc</font>
+
|<font face="Courier" size="+1">CTGACGTCCCTCTTCCGCGCCCCCTAAAATACCCA</font>
−
|align="center"|0.16
+
|align="center"|12.4
 +
|align="center"|0.187
 +
|align="center"|65.9
 
|-
 
|-
−
|align="center"|<partinfo>J23117</partinfo>
+
|align="center"|Col. 35
−
|<font face="Courier" size="+1">ttgacagctagctcagtcctagg<font color="red">g</font>att<font color="red">g</font>tgctagc</font>
+
|<font face="Courier" size="+1">CTGACGCCCAACCCGACACAGCGATATAATAATGA</font>
−
|align="center"|0.06
+
|align="center"|18.4
−
|-
+
|align="center"|0.89
−
|align="center"|<partinfo>J23118</partinfo>
+
|align="center"|20.7
−
|<font face="Courier" size="+1">ttgac<font color="red">g</font>gctagctcagtcctaggtatt<font color="red">g</font>tgctagc</font>
+
−
|align="center"|0.56
+
 
|-
 
|-
 +
 
|}
 
|}
  
 +
<html>
 +
<script>
 +
function more_color(){
 +
    var amino_string_containers = $("table font");
 +
    for(var i=0; i < amino_string_containers.length; i++){
 +
        var amino_string_container = amino_string_containers[i];
 +
        var amino_string = amino_string_container.textContent;
 +
        amino_string_container.textContent = "";
 +
        for(var j=0; j < amino_string.length; j++){
 +
            var letter = amino_string[j];
 +
            var color = "black";
 +
            //if( letter == "C"){
 +
            //    color = "green";
 +
            //} else if( letter == "A"){
 +
            //    color = "blue";
 +
            //} else if( letter == "T"){
 +
            //    color = "orange";
 +
            //} else if( letter == "G"){
 +
            //    color = "red";
 +
            //}
 +
            //$(amino_string_container).append("<font color=" + color + ">" + letter + "</font>");
 +
            if( letter == "C"){
 +
                color = "rgb(26,204,26)";
 +
            } else if( letter == "A"){
 +
                color = "rgb(128,179,230)";
 +
            } else if( letter == "T"){
 +
                color = "rgb(230,153,77)";
 +
            } else if( letter == "G"){
 +
                color = "rgb(230,128,128)";
 +
            }
 +
            $(amino_string_container).append('<font style="background-color: ' + color + '">' + letter + '</font>');
 +
        }
 +
    }
 +
}
 +
more_color();
 +
</script>
 +
</html>
  
 +
<sup>a</sup> The sequence of each pBAD promoter characterized. To use this simply produce a primer with the promoter sequence you wish in a tail. Replace the old promoter in your system e.g. [[Part:BBa_K808000]] with the new and see the chance. For more information go to ([http://2013.igem.org/Team:DTU-Denmark/pBAD_SPL link]).
  
 +
<sup>b</sup> Inducibility = Induced Strength divided by leakiness.
  
 +
=Characterization=
 +
We did a characterization by running growth experiments in parallel on a BioLector ([http://2013.igem.org/Team:DTU-Denmark/pBAD_SPL see more]). Below diagram sums up our results:
 +
[[File:690px-Induced vs basal.png|600px|center|Diagram over the promoter strength]]
  
−
 
+
=Conclusion=
−
 
+
The pBAD SPL contain promoters to the arabinose inducible system that will enable low leakiness but still maintain a reasonable promoter strength when turned on. This verifies that the SPL standard ([http://dspace.mit.edu/handle/1721.1/60080 RFC 63]) also can be used to produce promoters for inducible systems.
−
 
+
−
 
+
−
{| class="wikitable"
+
−
|-
+
−
! Mutant name !! Sequence
+
−
|-
+
−
| Mut. 1 || CTGACGACGCCCCTCTCCGCCCCTTAAAATGTCCA
+
−
|-
+
−
| Mut. 2 || CTGACGTAATCATACCGCCGAAAGTATTATCATTA
+
−
|-
+
−
| Mut. 3 || CTGACGCGACAACATTGCGTCCTATAAAATGCCGA
+
−
|}
+

Latest revision as of 22:06, 4 October 2013

Description

This is a page containing a synthetic promoter library (SPL) of pBAD promoters made by [http://2013.igem.org/Team:DTU-Denmark 2013 DTU iGEM team]. The library can be used to make and arabinose inducible system with very low leakiness, or tune the the promoter strength of the system when it's turned on.

A tight inducible system can be a very useful tool to express lethal or growth inhibiting proteins in E.coli. It can also be necessary with a non leaky expression system if you would like to visualize the expression of GFP SF in E.coli (see [http://2013.igem.org/Team:DTU-Denmark/HelloWorld link]).


The library

Here is a complete list of the sequences and their relative leakiness and strength:

Identifier Sequencea Induced Strength Leakiness Inducibilityb
Col. 02 CTGACGACGCCCCTCTCCGCCCCTTAAAATGTCCA 14.2 0.203 69.9
Col. 03 CTGACGTAATCATACCGCCGAAAGTATTATCATTA 16.6 0.426 39
Col. 04 CTGACGCGACAACATTGCGTCCTATAAAATGCCGA 14.2 0.345 41
Col. 05 CTGACGGCCCGCCCCCGATGCGCATAAAATACCAA 17.7 0.424 41.6
Col. 12 CTGACGCGTATCGCGAGCGGGCGTTATTATACGCA 13.6 0.414 32.9
Col. 10 CTGACGCCCCGAATCAGTAGTATTTATTATCTAGA 18 0.462 38.8
Col. 09 CTGACGCCACCCCCCCCCCCGGCGTATAATTCCCA 16.1 0.530 30.4
Col. 08 CTGACGCCCTACCGCTCGCCCCCCTATTATACCCA 13.4 0.211 63.4
Col. 13 CTGACGCCCCCCCCGTCCACCCCCTAATATCCCGA 10.9 0.096 113.6
Col. 15 CTGACGTGCGCCTGCCGTCCAAAGTAATATCCTTA 15.5 0.235 65.8
Col. 18 CTGACGCCCCGAATCAGTAGTATTTATTATCTAGA 19.8 0.524 37.8
Col. 19 CTGACGCTCGCACCGGCGACCCGATAATATCCATA 18.0 0.315 57
Col. 31 CTGACGCCCCCCCTCCCCCGACCTAAAATATCCAG 3.8 0.161 23.6
Col. 29 CTGACGCCGCTGCACCCGTCCCCCTATTATAGCAA 14.8 0.301 49.2
Col. 26 CTGACGCCGATCCTATCTCCCTATTAAAATTTTTA 13.8 0.292 47.2
Col. 22 CTGACGCCCCGAATCAGTAGTATTTATTATCTAGA 16.8 0.466 36.1
Col. 33 CTGACGCCAGCTTCCTACTTCAAATATAATCCGTA 14.4 1.01 14.3
Col. 34 CTGACGTCCCTCTTCCGCGCCCCCTAAAATACCCA 12.4 0.187 65.9
Col. 35 CTGACGCCCAACCCGACACAGCGATATAATAATGA 18.4 0.89 20.7

a The sequence of each pBAD promoter characterized. To use this simply produce a primer with the promoter sequence you wish in a tail. Replace the old promoter in your system e.g. Part:BBa_K808000 with the new and see the chance. For more information go to ([http://2013.igem.org/Team:DTU-Denmark/pBAD_SPL link]).

b Inducibility = Induced Strength divided by leakiness.

Characterization

We did a characterization by running growth experiments in parallel on a BioLector ([http://2013.igem.org/Team:DTU-Denmark/pBAD_SPL see more]). Below diagram sums up our results:

Diagram over the promoter strength

Conclusion

The pBAD SPL contain promoters to the arabinose inducible system that will enable low leakiness but still maintain a reasonable promoter strength when turned on. This verifies that the SPL standard ([http://dspace.mit.edu/handle/1721.1/60080 RFC 63]) also can be used to produce promoters for inducible systems.