Part:BBa_K3610038

BAK1 ectodomain / LargeBit for S. cerevisiae

This part contains the sequence for the ectodomain of the plant surface receptor BAK1 from A. thaliana fused to the LargeBit of the split NanoLuc system. Additionally, instead of the signal peptide native to the plant receptor, there is the secretion signal of the alpha factor from yeast at the N-terminal domain of the receptor, replacing the original signal sequence.

Usage and Biology

BAK1

The BRI1-associated receptor kinase (BAK1) is a leucin-rich repeat receptor kinase (LRR-RK) which interacts with multiple other LRR-RKs with different functions in hormone signalling and defense response. BAK1 localizes at the plasma membrane and the endosome. The BAK1 protein forms a structure with an extracellular domain with leucin-rich repeats, a single pass transmembrane domain and an intracellular domain with a kinase function.

Among others, BAK1 interacts with the LRR-RKs EF-Tu receptor (EFR), Flagellin sensing 2 (FLS2) and cold-shock protein receptor (CORE), all of which are pathogen recognition receptors (PRR) in brassicaceae plants. Upon binding of a microbe-associated molecular pattern at the LRR domain of the PRR, BAK1 forms a heterodimer with the PRR which triggers a phosphorylation cascade, leading to upregulation of defense mechanisms.

Usage with split-NanoLuc

In this case, the C-terminal domain of BAK1, entailing the intracellular kinase domain, was removed from the sequence. Instead, the LargeBit part of the split-NanoLuc protein domain was fused to the C-terminal domain via a 15 amino acid linker.

Interaction between the BAK1, which is a coreceptor to many other PRRs, is driven by the extracellular ligand-binding domain, further necessary is the transmembrane domain, including the juxtamembrane domain. Therefore, dimerization can be achieved without the intracellular kinase domain. Coexpressed with, for example, Part:BBa_K3610043, which is the PRR EFR that contains the N-terminal domain of the split-mCherry protein instead of the intracellular kinase domain, elf18-induced interaction between BAK1 and EFR is driving the reassembly of the LargeBit and the SmallBit of the NanoLuc luciferase, reconstituting its function to react with furimazine in the presence of oxygen, yielding furimamide and a fluorescent output. During our project, we expressed this part in S. cerevisiae in an attempt to visualize the ligand-dependent interaction between BAK1 and the respective plant PRR. This enables us to use this part, in coordination with different PRRs, to test for the presence of the epitopes which are recognized by the plant receptors.

Characterization

In our iGEM project, we designed a system to sense the presence of bacterial epitopes in water using this part.

We assembled this part with Golden Gate Cloning in a plasmid with a spectinomycin acetyltransferase, a selection marker, and amplified the plasmids in E. coli. The plasmids also contained the TRP1 gene. This encodes phosphoribosylanthranilate isomerase, an enzyme necessary for tryptophan synthesis. This made the same plasmids also suitable for expression in S. cerevisiae. We created plasmids containing this part with different promoters and we also transformed yeast cells with the different types of our plasmid. We had multiple versions of the ADH1 promoter from S. cerevisiae, one version was the full length promoter and the other one a truncated version.

This part was coexpressed in our yeast cells together with different parts, the parts Part:BBa_K3610043 and Part:BBa_K3610051, which encode for the EFR and the CORE receptor ectodomain respectively, with the ectodomains fused to the SmallBit part of the NanoBit system. These plasmids were obtained in the same manner, with the only difference that the selction marker for S. cerevisiae cells was not TRP1 but a gene for Kanamycin resistance.

After coexpressing the BAK1 ectodomain with either EFR of CORE in our yeast cells, a dimerization assay under a fluorometer was performed with a plate reader of the type Synergy H1.

Dimerization Assay

<tbody> UT</td><td>UT</td><td>UT + csp22</td><td>UT + csp22</td><td>UT + csp22</td><td>UT + csp22</td><td>UT + elf18</td><td>UT + elf18</td><td>UT + elf18</td>UT + elf18</td><td>CORE</td><td>CORE</td><td>CORE</td><td>CORE</td><td>CORE + csp22</td><td>CORE + csp22</td><td>CORE + csp22</td><td>CORE + csp22</td><td>CORE + elf18</td><td>CORE + elf18</td><td>CORE + elf18</td><td>CORE + elf18</td><td>EFR</td><td>EFR</td><td>EFR</td><td>EFR</td><td>EFR + csp22</td><td>EFR + csp22</td><td>EFR + csp22</td><td>EFR + csp22</td><td>EFR + elf18</td><td>EFR + elf18</td><td>EFR + elf18</td><td>EFR + elf18</td>

<tr><td>0.0009375</td><td>22.9</td><td>10</td><td>9</td>8</td><td>8</td><td>11</td><td>8</td><td>8</td><td>9</td><td>9</td><td>8</td><td>8</td>10</td><td>339</td><td>338</td><td>294</td><td>261</td><td>287</td><td>218</td><td>237</td><td>272</td><td>192</td><td>205</td><td>225</td><td>176</td><td>1279</td><td>1438</td><td>1524</td><td>1217</td><td>1160</td><td>1012</td><td>1071</td><td>1023</td><td>898</td><td>821</td><td>735</td><td>680</td></tr> <tr><td>0.00440972222222222</td><td>22.9</td><td>10</td><td>9</td>9</td><td>10</td><td>9</td><td>9</td><td>8</td><td>8</td><td>8</td><td>9</td><td>9</td>9</td><td>386</td><td>348</td><td>303</td><td>259</td><td>282</td><td>245</td><td>240</td><td>270</td><td>207</td><td>214</td><td>196</td><td>173</td><td>1258</td><td>1468</td><td>1409</td><td>1193</td><td>1079</td><td>955</td><td>958</td><td>973</td><td>931</td><td>840</td><td>739</td><td>718</td></tr> <tr><td>0.00788194444444444</td><td>23</td><td>8</td><td>9</td>10</td><td>8</td><td>8</td><td>9</td><td>8</td><td>10</td><td>8</td><td>8</td><td>8</td>8</td><td>392</td><td>379</td><td>302</td><td>296</td><td>309</td><td>239</td><td>264</td><td>271</td><td>209</td><td>204</td><td>199</td><td>168</td><td>1247</td><td>1369</td><td>1359</td><td>1160</td><td>992</td><td>869</td><td>932</td><td>933</td><td>956</td><td>914</td><td>814</td><td>760</td></tr> <tr><td>0.0113541666666667</td><td>23</td><td>11</td><td>8</td>9</td><td>9</td><td>8</td><td>10</td><td>8</td><td>9</td><td>9</td><td>9</td><td>9</td>9</td><td>409</td><td>357</td><td>325</td><td>301</td><td>307</td><td>241</td><td>265</td><td>254</td><td>214</td><td>199</td><td>197</td><td>171</td><td>1140</td><td>1314</td><td>1345</td><td>1120</td><td>979</td><td>860</td><td>1011</td><td>1010</td><td>969</td><td>901</td><td>853</td><td>801</td></tr> <tr><td>0.0148263888888889</td><td>23.1</td><td>9</td><td>9</td>9</td><td>10</td><td>10</td><td>10</td><td>8</td><td>8</td><td>9</td><td>8</td><td>10</td>9</td><td>401</td><td>375</td><td>305</td><td>300</td><td>295</td><td>253</td><td>244</td><td>279</td><td>216</td><td>194</td><td>207</td><td>166</td><td>1060</td><td>1234</td><td>1247</td><td>1072</td><td>993</td><td>871</td><td>1015</td><td>1037</td><td>955</td><td>889</td><td>806</td><td>812</td></tr> <tr><td>0.0182986111111111</td><td>23.1</td><td>9</td><td>11</td>9</td><td>10</td><td>9</td><td>9</td><td>9</td><td>9</td><td>9</td><td>9</td><td>8</td>8</td><td>398</td><td>395</td><td>318</td><td>273</td><td>295</td><td>238</td><td>254</td><td>256</td><td>212</td><td>203</td><td>183</td><td>169</td><td>997</td><td>1202</td><td>1199</td><td>1007</td><td>985</td><td>877</td><td>986</td><td>1003</td><td>911</td><td>876</td><td>827</td><td>821</td></tr> <tr><td>0.0217708333333333</td><td>23.1</td><td>8</td><td>9</td>9</td><td>8</td><td>9</td><td>9</td><td>9</td><td>9</td><td>8</td><td>9</td><td>8</td>9</td><td>404</td><td>369</td><td>310</td><td>294</td><td>303</td><td>238</td><td>267</td><td>230</td><td>204</td><td>192</td><td>165</td><td>157</td><td>964</td><td>1098</td><td>1077</td><td>943</td><td>988</td><td>843</td><td>970</td><td>978</td><td>865</td><td>859</td><td>808</td><td>824</td></tr> <tr><td>0.0252430555555556</td><td>23.2</td><td>10</td><td>10</td>9</td><td>8</td><td>8</td><td>8</td><td>9</td><td>8</td><td>8</td><td>9</td><td>9</td>8</td><td>405</td><td>387</td><td>290</td><td>282</td><td>290</td><td>239</td><td>232</td><td>231</td><td>194</td><td>196</td><td>175</td><td>160</td><td>906</td><td>1071</td><td>1061</td><td>909</td><td>946</td><td>834</td><td>952</td><td>984</td><td>861</td><td>816</td><td>795</td><td>809</td></tr> <tr><td>0.0287152777777778</td><td>23.2</td><td>9</td><td>8</td>9</td><td>9</td><td>9</td><td>9</td><td>9</td><td>8</td><td>8</td><td>11</td><td>8</td>8</td><td>407</td><td>380</td><td>270</td><td>292</td><td>304</td><td>239</td><td>256</td><td>228</td><td>203</td><td>188</td><td>161</td><td>146</td><td>883</td><td>1037</td><td>1023</td><td>895</td><td>882</td><td>824</td><td>923</td><td>968</td><td>803</td><td>843</td><td>789</td><td>795</td></tr> <tr><td>0.0321875</td><td>23.2</td><td>11</td><td>8</td>9</td><td>8</td><td>8</td><td>9</td><td>8</td><td>12</td><td>8</td><td>11</td><td>8</td>8</td><td>413</td><td>364</td><td>266</td><td>272</td><td>269</td><td>220</td><td>240</td><td>222</td><td>192</td><td>169</td><td>170</td><td>149</td><td>865</td><td>992</td><td>978</td><td>868</td><td>889</td><td>790</td><td>882</td><td>894</td><td>784</td><td>779</td><td>780</td><td>797</td></tr> <tr><td>0.0356597222222222</td><td>23.3</td><td>9</td><td>9</td>8</td><td>13</td><td>9</td><td>8</td><td>9</td><td>10</td><td>8</td><td>8</td><td>12</td>9</td><td>383</td><td>355</td><td>266</td><td>247</td><td>274</td><td>227</td><td>222</td><td>213</td><td>189</td><td>151</td><td>162</td><td>140</td><td>821</td><td>933</td><td>964</td><td>864</td><td>849</td><td>749</td><td>871</td><td>875</td><td>819</td><td>782</td><td>759</td><td>741</td></tr> <tr><td>0.0391319444444444</td><td>23.3</td><td>9</td><td>9</td>8</td><td>10</td><td>9</td><td>8</td><td>9</td><td>9</td><td>9</td><td>8</td><td>10</td>9</td><td>388</td><td>369</td><td>269</td><td>272</td><td>266</td><td>204</td><td>222</td><td>195</td><td>182</td><td>166</td><td>137</td><td>138</td><td>823</td><td>918</td><td>952</td><td>835</td><td>800</td><td>726</td><td>835</td><td>846</td><td>787</td><td>765</td><td>716</td><td>746</td></tr> <tr><td>0.0426041666666667</td><td>23.3</td><td>9</td><td>10</td>9</td><td>9</td><td>9</td><td>8</td><td>9</td><td>9</td><td>9</td><td>8</td><td>9</td>8</td><td>379</td><td>349</td><td>235</td><td>228</td><td>273</td><td>221</td><td>208</td><td>184</td><td>164</td><td>158</td><td>145</td><td>133</td><td>755</td><td>884</td><td>879</td><td>821</td><td>788</td><td>687</td><td>850</td><td>902</td><td>779</td><td>731</td><td>727</td><td>746</td></tr> <tr><td>0.0460763888888889</td><td>23.3</td><td>8</td><td>10</td>9</td><td>10</td><td>9</td><td>9</td><td>9</td><td>11</td><td>9</td><td>8</td><td>9</td>10</td><td>376</td><td>340</td><td>223</td><td>238</td><td>263</td><td>208</td><td>194</td><td>195</td><td>176</td><td>152</td><td>140</td><td>121</td><td>745</td><td>851</td><td>855</td><td>760</td><td>804</td><td>697</td><td>809</td><td>834</td><td>754</td><td>722</td><td>710</td><td>715</td></tr> <tr><td>0.0495486111111111</td><td>23.3</td><td>9</td><td>8</td>9</td><td>8</td><td>8</td><td>10</td><td>8</td><td>9</td><td>10</td><td>9</td><td>8</td>9</td><td>346</td><td>339</td><td>238</td><td>216</td><td>242</td><td>211</td><td>190</td><td>175</td><td>162</td><td>138</td><td>131</td><td>119</td><td>739</td><td>838</td><td>843</td><td>765</td><td>739</td><td>708</td><td>783</td><td>791</td><td>739</td><td>713</td><td>720</td><td>732</td></tr> <tr><td>0.0530208333333333</td><td>23.3</td><td>9</td><td>9</td>8</td><td>9</td><td>9</td><td>9</td><td>9</td><td>9</td><td>11</td><td>10</td><td>9</td>9</td><td>345</td><td>345</td><td>189</td><td>218</td><td>247</td><td>199</td><td>189</td><td>165</td><td>149</td><td>127</td><td>135</td><td>133</td><td>722</td><td>820</td><td>799</td><td>754</td><td>730</td><td>650</td><td>770</td><td>792</td><td>680</td><td>708</td><td>680</td><td>703</td></tr> <tr><td>0.0564930555555556</td><td>23.3</td><td>8</td><td>8</td>10</td><td>10</td><td>9</td><td>10</td><td>10</td><td>8</td><td>9</td><td>9</td><td>8</td>10</td><td>345</td><td>319</td><td>203</td><td>214</td><td>250</td><td>195</td><td>188</td><td>169</td><td>148</td><td>148</td><td>127</td><td>127</td><td>720</td><td>794</td><td>794</td><td>746</td><td>716</td><td>646</td><td>707</td><td>788</td><td>716</td><td>679</td><td>663</td><td>678</td></tr> <tr><td>0.0599652777777778</td><td>23.3</td><td>9</td><td>9</td>9</td><td>9</td><td>9</td><td>10</td><td>10</td><td>9</td><td>9</td><td>10</td><td>10</td>10</td><td>347</td><td>326</td><td>199</td><td>212</td><td>244</td><td>189</td><td>189</td><td>159</td><td>159</td><td>128</td><td>136</td><td>121</td><td>693</td><td>799</td><td>779</td><td>720</td><td>697</td><td>619</td><td>753</td><td>762</td><td>698</td><td>650</td><td>645</td><td>695</td></tr> <tr><td>0.0634375</td><td>23.3</td><td>9</td><td>9</td>9</td><td>9</td><td>9</td><td>8</td><td>9</td><td>9</td><td>9</td><td>11</td><td>9</td>9</td><td>326</td><td>309</td><td>188</td><td>216</td><td>225</td><td>187</td><td>180</td><td>157</td><td>140</td><td>117</td><td>125</td><td>113</td><td>659</td><td>775</td><td>738</td><td>709</td><td>702</td><td>607</td><td>726</td><td>737</td><td>699</td><td>642</td><td>630</td><td>670</td></tr> <tr><td>0.0669097222222222</td><td>23.3</td><td>9</td><td>9</td>9</td><td>8</td><td>9</td><td>9</td><td>9</td><td>9</td><td>9</td><td>8</td><td>8</td>8</td><td>347</td><td>295</td><td>170</td><td>197</td><td>224</td><td>180</td><td>168</td><td>157</td><td>148</td><td>114</td><td>109</td><td>100</td><td>668</td><td>722</td><td>733</td><td>678</td><td>664</td><td>580</td><td>666</td><td>700</td><td>633</td><td>655</td><td>598</td><td>647</td></tr> <tr><td>0.0703819444444444</td><td>23.3</td><td>9</td><td>10</td>10</td><td>9</td><td>10</td><td>9</td><td>8</td><td>9</td><td>9</td><td>9</td><td>10</td>10</td><td>313</td><td>298</td><td>178</td><td>186</td><td>225</td><td>189</td><td>159</td><td>148</td><td>133</td><td>117</td><td>123</td><td>97</td><td>635</td><td>683</td><td>699</td><td>652</td><td>651</td><td>577</td><td>679</td><td>729</td><td>647</td><td>604</td><td>585</td><td>607</td></tr> <tr><td>0.0738541666666667</td><td>23.3</td><td>9</td><td>8</td>9</td><td>10</td><td>8</td><td>8</td><td>10</td><td>8</td><td>9</td><td>10</td><td>10</td>11</td><td>302</td><td>293</td><td>160</td><td>181</td><td>220</td><td>170</td><td>167</td><td>144</td><td>130</td><td>120</td><td>113</td><td>109</td><td>660</td><td>713</td><td>701</td><td>622</td><td>640</td><td>574</td><td>657</td><td>650</td><td>621</td><td>607</td><td>579</td><td>606</td></tr> <tr><td>0.0773263888888889</td><td>23.3</td><td>11</td><td>10</td>9</td><td>9</td><td>9</td><td>9</td><td>9</td><td>8</td><td>9</td><td>10</td><td>8</td>9</td><td>296</td><td>283</td><td>164</td><td>160</td><td>216</td><td>172</td><td>149</td><td>145</td><td>119</td><td>98</td><td>113</td><td>106</td><td>603</td><td>676</td><td>662</td><td>610</td><td>624</td><td>564</td><td>641</td><td>673</td><td>607</td><td>582</td><td>550</td><td>597</td></tr> <tr><td>0.0807986111111111</td><td>23.3</td><td>9</td><td>8</td>8</td><td>10</td><td>9</td><td>8</td><td>9</td><td>10</td><td>9</td><td>10</td><td>10</td>10</td><td>283</td><td>257</td><td>147</td><td>161</td><td>194</td><td>159</td><td>139</td><td>138</td><td>123</td><td>108</td><td>115</td><td>108</td><td>609</td><td>656</td><td>660</td><td>610</td><td>594</td><td>540</td><td>651</td><td>625</td><td>590</td><td>564</td><td>550</td><td>584</td></tr> <tr><td>0.0842708333333333</td><td>23.3</td><td>8</td><td>9</td>9</td><td>9</td><td>9</td><td>9</td><td>9</td><td>10</td><td>9</td><td>11</td><td>9</td>9</td><td>282</td><td>264</td><td>150</td><td>169</td><td>178</td><td>162</td><td>141</td><td>133</td><td>119</td><td>94</td><td>118</td><td>102</td><td>598</td><td>652</td><td>626</td><td>627</td><td>586</td><td>524</td><td>618</td><td>630</td><td>575</td><td>561</td><td>551</td><td>598</td></tr> </table></tbody>

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 848
  Illegal PstI site found at 893
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 848
  Illegal PstI site found at 893
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1278
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 848
  Illegal PstI site found at 893
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 848
  Illegal PstI site found at 893
  
• 1000
  COMPATIBLE WITH RFC[1000]

[edit]

Categories

Parameters

Time

T° Lum

UT

UT

None