Latest revision as of 19:58, 17 October 2014

NRP UEA Norwich iGEM 2014

BioBrick Parts

This year the NRP-UEA iGEM team have submitted multiple parts to the iGEM registry which we hope will be extremely useful for not just future teams working on a plant synthetic biology project but also teams keen to experiment using Golden Gate technology. We have designed collections of plant specific parts and also improved the PSB1C3 plasmid to create a collection of parts generating a Golden Gate Modular Flipper to allow cloning in Golden Gate and subsequently submission to the registry in BioBrick standard.

New Parts

Bax (Bba_K1467203)
A reporter gene which, when transcribed, produces Bax protein that has a role in positively regulating cell apoptosis. The part has been made compatible with the GoldenGate MoClo Assembly Standard by removal of internal BsaI and BpiI recognition sequences.

Mannopine synthase (MAS) Promoter (Bba_K1467104)
A plant specific constitute promoter that has been made GoldenGate compatible by removal of internal BSAI and Bpi1 sites.

BS3 Promoter (Bba_K1467102)
A plant specific, TALE inducible promoter that has been made GoldenGate compatible by removal of internal BSAI and Bpi1 sites. The promoter becomes active in the presence of the TALE AvrBS3, driving the expression of coding regions.

PDF1.2 Promoter (Bba_K1467103)
A plant specific, Methyl Jasmonate inducible promoter that has been made GoldenGate compatible by removal of internal BSAI and Bpi1 sites. The promoter becomes active in the presence of methyl jasmonate (high methyl jasmonate levels indicate plant stress, often as a result of pathogen infection), driving the expression of coding regions.

AvrBS3 TALE (Bba_K1467205)
TAL (transcription activator-like) Effector that induces the BS3 promoter allowing safe testing of pathogen induced systems in the laboratory. The part has been made compatible with the GoldenGate MoClo Assembly Standard by removal of internal BsaI and BpiI recognition sequences.

Improved parts

RFP coding device - Golden Gate Module Flipper (Bba_K1467100)
A "GoldenGate Flipper" that can be used to convert Pro + 5U (promoter and 5' untranslated leader) modules from the GoldenGate MoClo Assembly Standard into standard BioBricks. It consists of the RFP reporter (BBa_J04450) flanked by an inverted pair of BsaI recognition sequences. GoldenGate MoClo parts can be flipped into BrioBrick parts in a one-pot, one-step, digestion-ligation GoldenGate cloning reaction.

RFP coding device - Golden Gate Module Flipper (Bba_K1467200)
A "GoldenGate Flipper" that can be used to convert CDS1 modules from the GoldenGate MoClo Assembly Standard into standard BioBricks. It consists of the RFP reporter (BBa_J04450) flanked by an inverted pair of BsaI recognition sequences. GoldenGate MoClo parts can be flipped into BrioBrick parts in a one-pot, one-step, digestion-ligation GoldenGate cloning reaction.

RFP coding device - Golden Gate Module Flipper (Bba_K1467300)
A "GoldenGate Flipper" that can be used to convert 3U + Ter (3' untranslated region and terminator) modules from the GoldenGate MoClo Assembly Standard into standard BioBricks. It consists of the RFP reporter (BBa_J04450) flanked by an inverted pair of BsaI recognition sequences. GoldenGate MoClo parts can be flipped into BrioBrick parts in a one-pot, one-step, digestion-ligation GoldenGate cloning reaction.

RFP coding device - Golden Gate Module Flipper (Bba_K1467400)
This part is a "GoldenGate Flipper" that can be used to assemble complete transcriptional units from GoldenGate MoClo Standard Parts producing a standard BioBrick. It consists of the RFP reporter (from BBa_J04450) flanked by an inverted pair of BsaI recognition sequences. GoldenGate MoClo parts can be flipped into BrioBrick parts in a one-pot, one-step, digestion-ligation GoldenGate cloning reaction.

35s Promoter (Bba_K1467101)
A plant specific constitutive promoter that has been made GoldenGate compatible by removal of internal BSAI and Bpi1 sites. The part was originally obtained from the Cauliflower Mosaic Virus and is intended for use as a constitutive promoter for gene expression experiments in plants. The part is an improvement on the part BBa_K414006.

AmilCP (Bba_K1467201)
A reporter chromoprotein which produces strong blue colour when expressed. A variation of the part BBa_K592009 that has been improved by removal of internal BsaI and BpiI recognition sequences- making it compatible with the GoldenGate MoClo Assembly Standard.

AmilGFP (Bba_K1467202)
A reporter chromoprotein which produces strong yellow colour when expressed. A variation of the part BBa_K592010 that has been improved by removal of internal BsaI and BpiI recognition sequences- making it compatible with the GoldenGate MoClo Assembly Standard.

GFP (Bba_K1467204)
A reporter protein that produces green fluorescence under UV light when expressed. A variation of the part BBa_E0040 that has been improved by removal of internal BsaI and BpiI recognition sequences- making it compatible with the GoldenGate MoClo Assembly Standard.

Plant specific parts

Mannopine synthase (MAS) Promoter (Bba_K1467104)
A plant specific constitute promoter that has been made GoldenGate compatible by removal of internal BSAI and Bpi1 sites.

BS3 Promoter (Bba_K1467102)
A plant specific, TALE inducible promoter that has been made GoldenGate compatible by removal of internal BSAI and Bpi1 sites. The promoter becomes active in the presence of the TALE AvrBS3, driving the expression of coding regions.

PDF1.2 Promoter (Bba_K1467103)
A plant specific, Methyl Jasmonate inducible promoter that has been made GoldenGate compatible by removal of internal BSAI and Bpi1 sites. The promoter becomes active in the presence of methyl jasmonate (high methyl jasmonate levels indicate plant stress, often as a result of pathogen infection), driving the expression of coding regions.

35s Promoter (Bba_K1467101)
A plant specific constitutive promoter that has been made GoldenGate compatible by removal of internal BSAI and Bpi1 sites. The part was originally obtained from the Cauliflower Mosaic Virus and is intended for use as a constitutive promoter for gene expression experiments in plants.

AvrBS3 TALE (Bba_K1467205)
TAL (transcription activator-like) Effector that induces the BS3 promoter allowing safe testing of pathogen induced systems in the laboratory. The part has been made compatible with the GoldenGate MoClo Assembly Standard by removal of internal BsaI and BpiI recognition sequences.

Compound parts

Our compound parts (e.g. Complete transcriptional units) had to be assembled in a plasmid backbone suitable for replication in Agrobacterium (as described on our System page), and so it was not possible to submit our compound parts to the Registry. Below is a list of the compound parts that we made and tested in plants. In collaboration with Valencia and Cambridge we are submitting an RCF for plants to allow future teams using plant chassis to do this.

Table 1: Level 1 compound parts

Promoter	Coding Sequence	Terminator	Infiltration Result
35s (BBa_K1467101)	GFP (BBa_K1467204)	ocs (octopine synthase)	GFP expression under UV light
BS3 (BBa_K1467102)	GFP (BBa_K1467204)	ocs (octopine synthase)	No GFP expression under UV light
PDF1.2 (BBa_K1467103)	GFP (BBa_K1467204)	ocs (octopine synthase)	Little GFP expression under UV light on untreated plant. GFP expression on treatment with Methyl Jasmonate
PR1	GFP (BBa_K1467204)	ocs (octopine synthase)	No GFP expression under UV light on untreated plant. GFP expression on treatment with Salicylic Acid
35s (BBa_K1467101)	Bax(BBa_K1467203)	ocs (octopine synthase)	Plant cell death
BS3 (BBa_K1467102)	Bax(BBa_K1467203)	ocs (octopine synthase)	-
MAS (BBa_K1467104)	AmilCP (BBa_K1467201)	ocs (octopine synthase)	-
MAS (BBa_K1467104)	AmilGFP(BBa_K1467202)	ocs (octopine synthase)	-
35s (BBa_K1467101)	AvrBS3(BBa_K1467205)	nos (nopaline synthase)	-

Table 2: Level 2 (Multigene) compound parts

Construct 1	Construct 2	Infiltration Result
BS3_GFP_ocs	35s_AvrBS3_nos	GFP expression under UV light
BS3_Bax_ocs	35s_AvrBS3_nos	-

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+<center>
+This year the NRP-UEA iGEM team have submitted multiple parts to the iGEM registry which we hope will be extremely useful for not just future teams working on a plant synthetic biology project but also teams keen to experiment using Golden Gate technology. We have designed collections of plant specific parts and also improved the PSB1C3 plasmid to create a collection of parts generating a Golden Gate Modular Flipper to allow cloning in Golden Gate and subsequently submission to the registry in BioBrick standard.
 <h3><u>New Parts</h3></u> <br>
 <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1467203" target="_blank">Bax (Bba_K1467203)</a><br>
 A reporter gene which, when transcribed, produces Bax protein that has a role in positively regulating cell apoptosis. The part has been made compatible with the GoldenGate MoClo Assembly Standard by removal of internal BsaI and BpiI recognition sequences.<br><br>
+<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1467104" target="_blank">Mannopine synthase (MAS) Promoter (Bba_K1467104)</a><br>
+A plant specific constitute promoter that has been made GoldenGate compatible by removal of internal BSAI and Bpi1 sites. <br><br>
+<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1467102" target="_blank">BS3 Promoter (Bba_K1467102)</a><br>
+A plant specific, TALE inducible promoter that has been made GoldenGate compatible by removal of internal BSAI and Bpi1 sites. The promoter becomes active in the presence of the TALE AvrBS3, driving the expression of coding regions.<br><br>
+<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1467103" target="_blank">PDF1.2 Promoter (Bba_K1467103)</a><br>
+A plant specific, Methyl Jasmonate inducible promoter that has been made GoldenGate compatible by removal of internal BSAI and Bpi1 sites. The promoter becomes active in the presence of methyl jasmonate (high methyl jasmonate levels indicate plant stress, often as a result of pathogen infection), driving the expression of coding regions.<br><br>
+<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1467205" target="_blank">AvrBS3 TALE (Bba_K1467205)</a><br>
+TAL (transcription activator-like) Effector that induces the BS3 promoter allowing safe testing of pathogen induced systems in the laboratory. The part has been made compatible with the GoldenGate MoClo Assembly Standard by removal of internal BsaI and BpiI recognition sequences.<br><br>
 <h3><u>Improved parts</h3></u><br>
-<a href="http://parts.igem.org/Part:BBa_K1467100">RFP coding device - Golden Gate Module Flipper (Bba_K1467100)</a><br>
+<a href="http://parts.igem.org/Part:BBa_K1467100" target="_blank">RFP coding device - Golden Gate Module Flipper (Bba_K1467100)</a><br>
 A "GoldenGate Flipper" that can be used to convert Pro + 5U (promoter and 5' untranslated leader) modules from the GoldenGate MoClo Assembly Standard into standard BioBricks. It consists of the RFP reporter (BBa_J04450) flanked by an inverted pair of BsaI recognition sequences. GoldenGate MoClo parts can be flipped into BrioBrick parts in a one-pot, one-step, digestion-ligation GoldenGate cloning reaction.<br><br>
-<a href="http://parts.igem.org/Part:BBa_K1467200">RFP coding device - Golden Gate Module Flipper (Bba_K1467200)</a><br>
+<a href="http://parts.igem.org/Part:BBa_K1467200" target="_blank">RFP coding device - Golden Gate Module Flipper (Bba_K1467200)</a><br>
 A "GoldenGate Flipper" that can be used to convert CDS1 modules from the GoldenGate MoClo Assembly Standard into standard BioBricks. It consists of the RFP reporter (BBa_J04450) flanked by an inverted pair of BsaI recognition sequences. GoldenGate MoClo parts can be flipped into BrioBrick parts in a one-pot, one-step, digestion-ligation GoldenGate cloning reaction.<br><br>
-<a href="http://parts.igem.org/Part:BBa_K1467300">RFP coding device - Golden Gate Module Flipper (Bba_K1467300)</a><br>
+<a href="http://parts.igem.org/Part:BBa_K1467300" target="_blank">RFP coding device - Golden Gate Module Flipper (Bba_K1467300)</a><br>
 A "GoldenGate Flipper" that can be used to convert 3U + Ter (3' untranslated region and terminator) modules from the GoldenGate MoClo Assembly Standard into standard BioBricks. It consists of the RFP reporter (BBa_J04450) flanked by an inverted pair of BsaI recognition sequences. GoldenGate MoClo parts can be flipped into BrioBrick parts in a one-pot, one-step, digestion-ligation GoldenGate cloning reaction.<br><br>
-<a href="http://parts.igem.org/Part:BBa_K1467400">RFP coding device - Golden Gate Module Flipper (Bba_K1467400)</a><br>
+<a href="http://parts.igem.org/Part:BBa_K1467400" target="_blank">RFP coding device - Golden Gate Module Flipper (Bba_K1467400)</a><br>
 This part is a "GoldenGate Flipper" that can be used to assemble complete transcriptional units from GoldenGate MoClo Standard Parts producing a standard BioBrick. It consists of the RFP reporter (from BBa_J04450) flanked by an inverted pair of BsaI recognition sequences. GoldenGate MoClo parts can be flipped into BrioBrick parts in a one-pot, one-step, digestion-ligation GoldenGate cloning reaction.<br><br>
+<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1467101" target="_blank">35s Promoter (Bba_K1467101)</a><br>
+A plant specific constitutive promoter that has been made GoldenGate compatible by removal of internal BSAI and Bpi1 sites. The part was originally obtained from the Cauliflower Mosaic Virus and is intended for use as a constitutive promoter for gene expression experiments in plants. The part is an improvement on the part BBa_K414006. <br><br>
 <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1467201" target="_blank">AmilCP (Bba_K1467201)</a><br>
-A reporter chromoprotein which produces strong blue colour when expressed. A variation of the part BBa_K592009 that has been improved by removal of internal BsaI and BpiI recognition sequences- making it compatible with the GoldenGate MoClo Assembly Standard and RFC 105.<br><br>
+A reporter chromoprotein which produces strong blue colour when expressed. A variation of the part BBa_K592009 that has been improved by removal of internal BsaI and BpiI recognition sequences- making it compatible with the GoldenGate MoClo Assembly Standard.<br><br>
 <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1467202" target="_blank">AmilGFP (Bba_K1467202)</a><br>
-A reporter chromoprotein which produces strong yellow colour when expressed. A variation of the part BBa_K592010 that has been improved by removal of internal BsaI and BpiI recognition sequences- making it compatible with the GoldenGate MoClo Assembly Standard and RFC 105.<br><br>
+A reporter chromoprotein which produces strong yellow colour when expressed. A variation of the part BBa_K592010 that has been improved by removal of internal BsaI and BpiI recognition sequences- making it compatible with the GoldenGate MoClo Assembly Standard.<br><br>
 <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1467204" target="_blank">GFP (Bba_K1467204)</a><br>
-A reporter protein that produces green fluorescence under UV light when expressed. A variation of the part BBa_E0040 that has been improved by removal of internal BsaI and BpiI recognition sequences- making it compatible with the GoldenGate MoClo Assembly Standard and RFC 105.<br><br>
+A reporter protein that produces green fluorescence under UV light when expressed. A variation of the part BBa_E0040 that has been improved by removal of internal BsaI and BpiI recognition sequences- making it compatible with the GoldenGate MoClo Assembly Standard.<br><br>
@@ Line 162: / Line 180: @@
 TAL (transcription activator-like) Effector that induces the BS3 promoter allowing safe testing of pathogen induced systems in the laboratory. The part has been made compatible with the GoldenGate MoClo Assembly Standard by removal of internal BsaI and BpiI recognition sequences.<br><br>
+<h3><u>Compound parts</h3></u><br>
+Our compound parts (e.g. Complete transcriptional units) had to be assembled in a plasmid backbone suitable for replication in Agrobacterium (as described on our <a href="https://2014.igem.org/Team:NRP-UEA-Norwich/Project_System">System page</a>), and so it was not possible to submit our compound parts to the Registry.  Below is a list of the compound parts that we made and tested in plants. In <a href="https://2014.igem.org/Team:NRP-UEA-Norwich/HP_Collaborations">collaboration with Valencia and Cambridge</a> we are submitting an RCF for plants to allow future teams using plant chassis to do this. <br><br>
+<u>Table 1: Level 1 compound parts</u><br><br>
+<table width="70%" border="1" summary="Bronze" align="center" cellspacing="10" cellpadding="10">
+<col width="300">
+<col width="300">
+<col width="300">
+<col width="300">
+ <tr>
+  <th>Promoter</th>
+  <th>Coding Sequence</th>
+ <th>Terminator</th>
+   <th>Infiltration Result</th>
+ </tr>
+ <tr>
+  <td>35s (BBa_K1467101)</td>
+<td>GFP (BBa_K1467204)</td>
+<td>ocs (octopine synthase)</td>
+  <td> <a href="https://static.igem.org/mediawiki/parts/d/da/35s_GFP.jpg"> GFP expression under UV light</a></td>
+ </tr>
+ <tr>
+  <td>BS3 (BBa_K1467102)</td>
+ <td>GFP (BBa_K1467204)</td>
+ <td>ocs (octopine synthase)</td>
+  <td>No GFP expression under UV light</td>
+ </tr>
+ <tr>
+  <td>PDF1.2 (BBa_K1467103)</td>
+ <td>GFP (BBa_K1467204)</td>
+ <td>ocs (octopine synthase)</td>
+  <td><a href="https://static.igem.org/mediawiki/parts/0/0d/Image-_PDF1.2_GFP.jpg"> Little GFP expression under UV light on untreated plant. GFP expression on treatment with Methyl Jasmonate </a> </td>
+ </tr>
+ <tr>
+  <td>PR1</td>
+ <td>GFP (BBa_K1467204)</td>
+ <td>ocs (octopine synthase)</td>
+  <td>No GFP expression under UV light on untreated plant. GFP expression on treatment with Salicylic Acid </td>
+ </tr>
+  <tr>
+  <td>35s (BBa_K1467101)</td>
+ <td> Bax(BBa_K1467203)</td>
+ <td>ocs (octopine synthase)</td>
+  <td> <a href="https://static.igem.org/mediawiki/parts/8/8b/35s_Bax_plus_control.jpg"> Plant cell death </a> </td>
+ </tr>
+ <tr>
+  <td>BS3 (BBa_K1467102)</td>
+ <td> Bax(BBa_K1467203)</td>
+ <td>ocs (octopine synthase)</td>
+  <td> - </td>
+ </tr>
+<tr>
+  <td>MAS (BBa_K1467104)</td>
+ <td>AmilCP (BBa_K1467201)</td>
+ <td>ocs (octopine synthase)</td>
+  <td>-</td>
+ </tr>
+<tr>
+  <td>MAS (BBa_K1467104)</td>
+ <td>AmilGFP(BBa_K1467202)</td>
+ <td>ocs (octopine synthase)</td>
+  <td>-</td>
+ </tr>
+<tr>
+  <td>35s (BBa_K1467101)</td>
+ <td>AvrBS3(BBa_K1467205)</td>
+ <td>nos (nopaline synthase)</td>
+  <td>-</td>
+ </tr>
+</table><br><br>
+<u>Table 2: Level 2 (Multigene) compound parts</u><br><br>
+<table width="70%" border="1" summary="Bronze" align="center" cellspacing="10" cellpadding="10">
+<col width="300">
+<col width="300">
+<col width="300">
+ <tr>
+  <th>Construct 1</th>
+  <th>Construct 2</th>
+  <th>Infiltration Result</th>
+ </tr>
+ <tr>
+  <td>BS3_GFP_ocs</td>
+<td>35s_AvrBS3_nos</td>
+  <td> <a href="https://static.igem.org/mediawiki/parts/7/77/BS3_GFP_%2B_AvrBS3.jpg"> GFP expression under UV light</a></td>
+ </tr>
+<tr>
+  <td>BS3_Bax_ocs</td>
+<td>35s_AvrBS3_nos</td>
+  <td> - </a></td>
+ </tr>
+</table><br><br>
+<center>
        </div>

Team:NRP-UEA-Norwich/Project Parts

From 2014.igem.org