Team:Manchester/Parts

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We submitted and sequenced three biobricks integral to our project. These can be found below:

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This year we submitted and characterised 3 BioBricks, one of which is an improvement of BBa_K925000! Characterisation below:

Successful Expression of delta 9 and delta 12 desaturase, and FabA

In order to characterise our biobricks, we made use of the standard parts found in the registry. By inserting the created biobricks BBa_K1027001 and BBa_K1027002 in to BBa_K608002 (a biobrick consisting of a ribosomal binding site and a constitutive promoter), we were able to create a new construct that expressed the delta 9 desaturase and delta 12 desaturase proteins. The constructs are shown above.

Plates

Another piece of evidence suggesting that our constructs were successfully expressing our delta 9 desaturase and delta 12 desaturase enzymes was the size of the colonies grown on the plates. Pictures of the plates are shown below. On the far left is a control plate, with bacteria transformed with BBa_K608002 but with no gene inserted in front of the promoter. To the right of that are our delta 12 colonies. They are much smaller than the control, and even taking 20 hours to grow to that size as opposed to the usual 16 hours. We hypothesise that because delta 12 desaturase is a membrane-bound protein that the E. coli does not normally express, constitutively expressing it could inhibit the bacteria and slow growth. Compare this to fabA on the far right. Looking through the literature, we found that overexpression of fabA results in no significant difference in growth size or speed relative to the wild type strain (Luo et al, 2009). Compared to the control plate, we also found that the colonies grew normally.

Gel Digests

To confirm that our desired genes were in fact within the expression construct mentioned above, we carried out test digests of our ligated plasmids. Happily, when we ran our digests on an agarose gel, we saw all of the bands we would predict from the expected fragments. The gel pictures are on the right.
delta 9 desaturase: Cut with BamHI + EcoRV. Expected bands 1424 bp, 1262 bp, 99 bp.
delta 12 desaturase: Cut with BamHI, XbaI, PstI. Expected bands 2127 bp, 656 bp, 430 bp.
FabA Cut with EcoRV. Expected bands 1429 bp, 1115 bp. Cut with EcoRV, PstI. Expected bands 1153 bp, 1115 bp, 339 bp.

Orbitrap LC-MS analysis
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Summary
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              <p><b><a id="Q1">Successful Expression of delta 9 and delta 12 desaturase, and FabA</a></b><br>
 <p>In order to characterise our biobricks, we made use of the standard parts found in the registry. By inserting the created biobricks BBa_K1027001 and BBa_K1027002 in to BBa_K608002 (a biobrick consisting of a ribosomal binding site and a constitutive promoter), we were able to create a new construct that expressed the delta 9 desaturase and delta 12 desaturase proteins. The constructs are shown above.</p>
-<p><b>Plates</b></p>
-<p>Another piece of evidence suggesting that our constructs were successfully expressing our delta 9 desaturase and delta 12 desaturase enzymes was the size of the colonies grown on the plates. Pictures of the plates are shown below. On the far left is a control plate, with bacteria transformed with BBa_K608002 but with no gene inserted in front of the promoter. To the right of that are our delta 12 colonies. They are much smaller than the control, and even taking 20 hours to grow to that size as opposed to the usual 16 hours. We hypothesise that because delta 12 desaturase is a membrane-bound protein that the <i>E. coli</i> does not normally express, constitutively expressing it could inhibit the bacteria and slow growth. Compare this to fabA on the far right. Looking through the literature, we found that overexpression of fabA results in no significant difference in growth size or speed relative to the wild type strain (Luo et al, 2009). Compared to the control plate, we also found that the colonies grew normally.</p>
-<p><b>Gel Digests</b></p>
-<p>To confirm that our desired genes were in fact within the expression construct mentioned above, we carried out test digests of our ligated plasmids. Happily, when we ran our digests on an agarose gel, we saw all of the bands we would predict from the expected fragments. The gel pictures are on the right. <br>
-<p>In order to characterise our biobricks, we made use of the standard parts found in the registry. By inserting the created biobricks BBa_K1027001 and BBa_K1027002 in to BBa_K608002 (a biobrick consisting of a ribosomal binding site and a constitutive promoter), we were able to create a new construct that expressed the delta 9 desaturase and delta 12 desaturase proteins. The constructs are shown above.</p>
-<p><b>Plates</b></p>
-<p>Another piece of evidence suggesting that our constructs were successfully expressing our delta 9 desaturase and delta 12 desaturase enzymes was the size of the colonies grown on the plates. Pictures of the plates are shown below. On the far left is a control plate, with bacteria transformed with BBa_K608002 but with no gene inserted in front of the promoter. To the right of that are our delta 12 colonies. They are much smaller than the control, and even taking 20 hours to grow to that size as opposed to the usual 16 hours. We hypothesise that because delta 12 desaturase is a membrane-bound protein that the <i>E. coli</i> does not normally express, constitutively expressing it could inhibit the bacteria and slow growth. Compare this to fabA on the far right. Looking through the literature, we found that overexpression of fabA results in no significant difference in growth size or speed relative to the wild type strain (Luo et al, 2009). Compared to the control plate, we also found that the colonies grew normally.</p>
-<p><b>Gel Digests</b></p>
-<p>To confirm that our desired genes were in fact within the expression construct mentioned above, we carried out test digests of our ligated plasmids. Happily, when we ran our digests on an agarose gel, we saw all of the bands we would predict from the expected fragments. The gel pictures are on the right. <br>
-<b>delta 9 desaturase:</b> Cut with x, x, x. Expected bands x, x, x.<br>
-<b>delta 12 desaturase:</b> Cut with x, x, x. Expected bands x, x, x.<br>
-<b>FabA</b> Cut with x, x, x. Expected bands x, x, x.<br></p><p>In order to characterise our biobricks, we made use of the standard parts found in the registry. By inserting the created biobricks BBa_K1027001 and BBa_K1027002 in to BBa_K608002 (a biobrick consisting of a ribosomal binding site and a constitutive promoter), we were able to create a new construct that expressed the delta 9 desaturase and delta 12 desaturase proteins. The constructs are shown above.</p>
-<p><b>Plates</b></p>
-<p>Another piece of evidence suggesting that our constructs were successfully expressing our delta 9 desaturase and delta 12 desaturase enzymes was the size of the colonies grown on the plates. Pictures of the plates are shown below. On the far left is a control plate, with bacteria transformed with BBa_K608002 but with no gene inserted in front of the promoter. To the right of that are our delta 12 colonies. They are much smaller than the control, and even taking 20 hours to grow to that size as opposed to the usual 16 hours. We hypothesise that because delta 12 desaturase is a membrane-bound protein that the <i>E. coli</i> does not normally express, constitutively expressing it could inhibit the bacteria and slow growth. Compare this to fabA on the far right. Looking through the literature, we found that overexpression of fabA results in no significant difference in growth size or speed relative to the wild type strain (Luo et al, 2009). Compared to the control plate, we also found that the colonies grew normally.</p>
-<p><b>Gel Digests</b></p>
-<p>To confirm that our desired genes were in fact within the expression construct mentioned above, we carried out test digests of our ligated plasmids. Happily, when we ran our digests on an agarose gel, we saw all of the bands we would predict from the expected fragments. The gel pictures are on the right. <br>
-<b>delta 9 desaturase:</b> Cut with x, x, x. Expected bands x, x, x.<br>
-<b>delta 12 desaturase:</b> Cut with x, x, x. Expected bands x, x, x.<br>
-<b>FabA</b> Cut with x, x, x. Expected bands x, x, x.<br></p><p>In order to characterise our biobricks, we made use of the standard parts found in the registry. By inserting the created biobricks BBa_K1027001 and BBa_K1027002 in to BBa_K608002 (a biobrick consisting of a ribosomal binding site and a constitutive promoter), we were able to create a new construct that expressed the delta 9 desaturase and delta 12 desaturase proteins. The constructs are shown above.</p>
-<p><b>Plates</b></p>
-<p>Another piece of evidence suggesting that our constructs were successfully expressing our delta 9 desaturase and delta 12 desaturase enzymes was the size of the colonies grown on the plates. Pictures of the plates are shown below. On the far left is a control plate, with bacteria transformed with BBa_K608002 but with no gene inserted in front of the promoter. To the right of that are our delta 12 colonies. They are much smaller than the control, and even taking 20 hours to grow to that size as opposed to the usual 16 hours. We hypothesise that because delta 12 desaturase is a membrane-bound protein that the <i>E. coli</i> does not normally express, constitutively expressing it could inhibit the bacteria and slow growth. Compare this to fabA on the far right. Looking through the literature, we found that overexpression of fabA results in no significant difference in growth size or speed relative to the wild type strain (Luo et al, 2009). Compared to the control plate, we also found that the colonies grew normally.</p>
-<p><b>Gel Digests</b></p>
-<p>To confirm that our desired genes were in fact within the expression construct mentioned above, we carried out test digests of our ligated plasmids. Happily, when we ran our digests on an agarose gel, we saw all of the bands we would predict from the expected fragments. The gel pictures are on the right. <br>
-<b>delta 9 desaturase:</b> Cut with x, x, x. Expected bands x, x, x.<br>
-<b>delta 12 desaturase:</b> Cut with x, x, x. Expected bands x, x, x.<br>
-<b>FabA</b> Cut with x, x, x. Expected bands x, x, x.<br></p><p>In order to characterise our biobricks, we made use of the standard parts found in the registry. By inserting the created biobricks BBa_K1027001 and BBa_K1027002 in to BBa_K608002 (a biobrick consisting of a ribosomal binding site and a constitutive promoter), we were able to create a new construct that expressed the delta 9 desaturase and delta 12 desaturase proteins. The constructs are shown above.</p>
 <p><b>Plates</b></p>