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Template:Team:Uppsala/JS/notebook
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| - | + | ds = '<div id="dairy-text"><h1>Saturday 2013-08-24</h1><br><b>Name of participants: </b>Mikael Strandgren, Anders Berglund, Alexander, Pontus, Sabri, Magnus, Marcus H, Karl H, Hampus.E, Lovisa P, Kristoffer L, Emil M<br><br><h2>Ongoing constructs:</h2><br><b>Escherichia coli</b><br>257. pSB1C3-pemp<br>288. pSB1C3-pem<br>136. pSB1C3-B0034-USP<br>pSB1C3-B0034-usp45<br>102.pSB1C3-CrtE<br>108.pEL3S17-J23106-B0034-IspA<br>109.pEL3S17-K206000-B0034-IspA<br>110.pEL3S17-K206001-B0034-IspA<br>140.pEL3S17-J23110-B0034-UGTCs2-6xHis<br>141.pEL3S17-J23101-B0034-UGTCs2-6xHis<br>142.pEL3S17-CP1-B0034-UGTCs2-6xHis<br>143.pEL3S17-CP8-B0034-UGTCs2-6xHis<br>147.pSB1C3-B0034-Thioredoxin<br>163.pEL3S17-ZCD-His-B0034-UGTCs2-His<br>177.pEL3S17-J23101-B0034-Exp-Linker-Miraculin-His<br>178.pEL3S17-J23110-B0034-Exp-Linker-Miraculin-His<br>179.pEL3S17-CP1-B0034-Exp-Linker-Miraculin-His<br>180.pEL3S17-CP8-B0034-Exp-Linker-Miraculin-His<br>181.pSBLb4C15-CP1-B0034-Exp-Linker-Miraculin-His<br>182.pSBLb4C15-CP8-B0034-Exp-Linker-Miraculin-His<br>183.pEL3A15-B0034-CrtZ<br>184.pSB1C3-B0034-CrtZ<br>(201. E.Coli. Nissle - CrtY-CrtI)<br>202.pSB4C5-CrtE-B0034-Blues-CrtB-B0034-CrtI-Linker-Zif268<br>203.pSB1C3-CrtE-B0034-Blues-Linker-CrtB-B0034-CrtI-Linker-Zif268-B0034-PBSII-Linker-CrtY-B0034-CrtZ-Linker-Gli1<br>208. pSB1A3-B0034-Thioredoxin-ZCD-His<br>213.pSB1K3-pBAD/AraC-B0034-Jazz-Linker-CrtE-B0034-Blues-Linker-CrtB-B0034-CrtI-Linker-Zif268-B0034-PBSII-Linker-CrtY-B0034-HivC-Linker-CrtO-B0034-CrtZ-Linker-Gli1<br>214.pEL3K16-pBAD/AraC-B0034-Jazz-Linker-CrtE-B0034-Blues-Linker-CrtB-B0034-CrtI-Linker-Zif268-B0034-PBSII-Linker-CrtY-B0034-HivC-Linker-CrtO-B0034-CrtZ-Linker-Gli1<br>215.pSB1K3-pBAD/AraC-B0034-Jazz-Linker-CrtE-B0034-Blues-Linker-CrtB-B0034-CrtI-Linker-Zif268-B0034-PBSII-Linker-CrtY-B0034-CrtZ-Linker-Gli1<br>216.pEL3K16-pBAD/AraC-B0034-Jazz-Linker-CrtE-B0034-Blues-Linker-CrtB-B0034-CrtI-Linker-Zif268-B0034-PBSII-Linker-CrtY-B0034-CrtZ-Linker-Gli1<br>217.pSB3K16-pBAD/AraC-B0034-Jazz-Linker-CrtE-B0034-Blues-Linker-CrtB-B0034-CrtI-Linker-Zif268<br>218.pEL3K16-pBAD/AraC-B0034-Jazz-Linker-CrtE-B0034-Blues-Linker-CrtB-B0034-CrtI-Linker-Zif268<br>220.pSB3K3-J23110-B0034-UGTCs2-His<br>221.pSB3T5-J23101-B0034-UGTCs2-His<br>222.pSB3K3-CP1-B0034-UGTCs2-His<br>223.pSB3K3-CP8-B0034-UGTCs2-His<br>264.pSB3K3-J23101-B0034-Exp-Linker-Miraculin-His<br>265.psB3K3-J23110-B0034-Exp-Linker-Miraculin-His<br>266.psB3K3-CP1-B0034-Exp-Linker-Miraculin-His<br>269.psB3K3-CP8-B0034-Exp-Linker-Miraculin-His<br>272. pSB1K3-B0034-Thioredoxin-ZCD-His<br>299. pEL3S17-J23101-B0034-Thioredoxin<br>300. pEL3S17-J23106-B0034-Thioredoxin<br>301. pEL3S17-J23110-B0034-Thioredoxin<br>303. pSB1A3-j23101-B0034-CrtZ<br>304. pSB1A3-j23106-B0034-CrtZ<br>305.pSB1K3-J23110-CrtZ<br>308.pSB1C3-Miraculin<br>309.pSB1C3-B0034-Miraculin<br>261. pSB3K3-J23110-B0034-His-4CL<br>333. pSB3K3-J23110-HIS-STS<br>334. pSB3K3-CP1-His-STS<br>335. pSB3K3-J23101-HIs-STS<br><br><b>Lactobacillus reuteri:</b><br>Lb35. reuteri DSM 20016 pVS2<br>Lb36. reuteri DSM 20016 pSBLB4B15-CP30-mCherry<br>Lb37. reuteri DSM 20016 pSBLB4B15-CP30-amilGFP<br>Lb38. reuteri DSM 20016 pSBLB4B15-CP11-amilCP<br>Lb39. reuteri DSM 20016 pSBLB4B15-CP30-amilCP<br>Lb40. reuteri DSM 20016 pSBLB4C15-CP11-aeBlue<br>Lb41. reuteri DSM 20016 pSBLB4C15-CP30-aeBlue<br>Lb42. reuteri DSM 20016 pSBLB4C15-CP1-BFP<br>Lb43. reuteri DSM 20016 pSBLB4C15-CP8-BFP<br>Lb44. reuteri DSM 20016 pSBLB4C15-CP11-BFP<br>Lb45. reuteri DSM 20016 pSBLB4C15-CP29-BFP<br>Lb46. reuteri DSM 20016 pSBLB4C15-CP30-BFP<br><br><b>Lactobacillus plantarum:</b><br>Lb31. plantarum 36E pVS2<br>Lb32. plantarum 36E pSBLB4B15-CP30-fusionGFP<br>Lb33. plantarum 36E pSBLB4B15-CP11-usp45-fusionGFP<br>Lb34. plantarum 36E pSBLB4B15-CP11-tsPurple<br><br><h2>Todays work:</h2><br><b>Mutagenesis</b><br>338.pSB1C3-meffBlue <br><br><b>Digest:</b><br>200.pSB1C3-B0034-asCP E,X<br>148.pSB1C3-RBS-gfasPurple E,X<br>20.pBAD/AraC-B0034-Jazz-Linker-CrtE-B0034-Blues-Linker-CrtB-B0034-CrtI-Linker-Zif268-B0034-PBSII-Linker-CrtY-B0034-CrtZ-Linker-Gli1 E,X<br>257. pSB1C3-pemp (two tubes) with Pem-mutE_F & Pem-mutE_R (EcoRI)<br>288. pSB1C3-pem (two tubes) with Pem-mutE_F & Pem-mutE_R (EcoRI)<br><br><b>Ligation:</b><br>200.pSB1C3-B0034-asCP+91.BBa_J61002 (amp)-J23106→<br>→ 343.J23106-B0034-asCP<br>148.pSB1C3-RBS-gfasPurple+91.BBa_J61002 (amp)-J23106→<br>→ 160.j23106-RBS-gfasPurple<br>148.pSB1C3-RBS-gfasPurple+92.BBa_J61002 (amp)-J23110→<br>→ 159j23110-RBS-gfasPurple<br>20.pSB4C5-pBAD/AraC-B0034-Jazz-Linker-CrtE-B0034-Blues-Linker-CrtB-B0034-CrtI-Linker-Zif268-B0034-PBSII-Linker-CrtY-B0034-CrtZ-Linker-Gli1+6.pSB1K3-red→<br>→215.pSB1K3-pSB1K3-pBAD/AraC-B0034-Jazz-Linker-CrtE-B0034-Blues-Linker-CrtB-B0034-CrtI-Linker-Zif268-B0034-PBSII-Linker-CrtY-B0034-CrtZ-Linker-Gli1<br>184.pSB1C3-B0034-CrtZ+6.pSB1K3-Red+103.pSB1K3-J23101→<br>→ 303.pSB1K3-J23101-B0034-CrtZ<br>127.pSB1A3-B0034-UGTCs2-His+7.pSB3K3-Red+CP8→<br>→ 223.pSB3K3-CP8-B0034-UGTCs2-His<br><br><b>Transformation:</b><br>223.pSB3K3-CP8-B0034-UGTCs2-His<br>163.pEL3S17-ZCD-His-B0034-UGTCs2-His<br>182.pSBLb4C15-CP8-B0034-Exp-Linker-Miraculin-His<br>208.pSB1A3-B0034-Thioredoxin-ZCD-His<br>215.pSB1K3-pBAD/AraC-B0034-Jazz-Linker-CrtE-B0034-Blues-Linker-CrtB-B0034-CrtI-Linker-Zif268-B0034-PBSII-Linker-CrtY-B0034-CrtZ-Linker-Gli1<br>343.pSB1C3-J23106-B0034-asCP<br>160.pSB1C3-j23106-RBS-gfasPurple<br>159.pSB1C3-j23110-RBS-gfasPurple<br>303.pSB1K3-j23101-B0034-CrtZ<br>269.pSB3K3-CP8-B0034-Exp-Linker-Miraculin-His<br>336.pSB1C3.spisPink<br>337.pSB1C3.scOrange<br>284.pSB1C3.B0034-aeBlue<br>285.pSB1C3.J23110-B0034-aeBlue<br>286.pSB1C3.J23110-B0034-amilCP<br>287.pSB1C3-J23110-B0034-asCP<br>336.pSB1C3-spisPink<br>337.pSB1C3-scOrange<br>284. pSB1C3-B0034-aeBlue<br>285. pSB1C3-J23110-B0034-aeBlue<br>286. pSB1C3-J23110-B0034-amilCP<br>287. pSB1C3-J23110-B0034-asCP<br><br><b>O/N screening:</b><br>339. pSB1C3-gfasPurple<br>340. pSB1C3-amajCP(amajLime)<br>341. pSB1C3-tsPurple<br>342. pSB1C3-fwYellow<br>Lb31. plantarum 36E pVS2<br>Lb32. plantarum 36E pSBLB4B15-CP30-fusionGFP<br>Lb33. plantarum 36E pSBLB4B15-CP11-usp45-fusionGFP<br>Lb34. plantarum 36E pSBLB4B15-CP11-tsPurple<br>Lb35. reuteri DSM 20016 pVS2<br>Lb36. reuteri DSM 20016 pSBLB4B15-CP30-mCherry<br>Lb37. reuteri DSM 20016 pSBLB4B15-CP30-amilGFP<br>Lb38. reuteri DSM 20016 pSBLB4B15-CP11-amilCP<br>Lb39. reuteri DSM 20016 pSBLB4B15-CP30-amilCP<br>Lb40. reuteri DSM 20016 pSBLB4C15-CP11-aeBlue<br>Lb41. reuteri DSM 20016 pSBLB4C15-CP30-aeBlue<br>Lb42. reuteri DSM 20016 pSBLB4C15-CP1-BFP<br>Lb43. reuteri DSM 20016 pSBLB4C15-CP8-BFP<br>Lb44. reuteri DSM 20016 pSBLB4C15-CP11-BFP<br>Lb45. reuteri DSM 20016 pSBLB4C15-CP29-BFP<br>Lb46. reuteri DSM 20016 pSBLB4C15-CP30-BFP<br>136.<br>312<br>313<br>315<br>321<br>325<br>327<br>332<br>317<br><br><b>O/N:</b><br>247. pSB1C3-J23110-RBS-tsPurple<br>248. pSB1C3-J23106-RBS-tsPurple (grow 30 degrees)<br>249. pSB1C3-J23110-RBS-scOrange<br>250. pSB1C3-J23106-RBS-scOrange (grow 30 degrees)<br>251. pSB1C3-J23110-RBS-fwYellow<br>252. pSB1C3-J23106-RBS-fwYellow (grow 30 degrees)<br>143. pEL3S17-CP8-B0034-UGTCs2-6xHis<br>179. pEL3S17-CP1-B0034-Exp-Linker-Miraculin-His<br>180. pEL3S17-CP8-B0034-Exp-Linker-Miraculin-His<br>265. J23110-B0034-Exp-Linker-Miraculin-His<br>301. pEL3S17-J23110-B0034-Thioredoxin<br><br><b>Plasmid prepp & sequence prepp</b><br>155. pSB1C3-j23110-RBS-meffRed<br>156. pSB1C3-j23106-RBS-meffRed<br>157. pSB1C3-j23110-RBS-spisPink<br>158. pSB1C3-j23106-RBS-spisPink<br><br><b>Gel electrophoresis:</b><br>Lb31. plantarum 36E pVS2<br>Lb32. plantarum 36E pSBLB4B15-CP30-fusionGFP<br>Lb33. plantarum 36E pSBLB4B15-CP11-usp45-fusionGFP<br>Lb34. plantarum 36E pSBLB4B15-CP11-tsPurple<br>Lb35. reuteri DSM 20016 pVS2<br>Lb36. reuteri DSM 20016 pSBLB4B15-CP30-mCherry<br>Lb37. reuteri DSM 20016 pSBLB4B15-CP30-amilGFP<br>Lb38. reuteri DSM 20016 pSBLB4B15-CP11-amilCP<br>Lb39. reuteri DSM 20016 pSBLB4B15-CP30-amilCP<br>Lb40. reuteri DSM 20016 pSBLB4C15-CP11-aeBlue<br>Lb41. reuteri DSM 20016 pSBLB4C15-CP30-aeBlue<br>Lb42. reuteri DSM 20016 pSBLB4C15-CP1-BFP<br>Lb43. reuteri DSM 20016 pSBLB4C15-CP8-BFP<br>Lb44. reuteri DSM 20016 pSBLB4C15-CP11-BFP<br>Lb45. reuteri DSM 20016 pSBLB4C15-CP29-BFP<br>Lb46. reuteri DSM 20016 pSBLB4C15-CP30-BFP<br>257. pSB1C3-pemp (two tubes) with Pem-mutE_F & Pem-mutE_R<br>288. pSB1C3-pem (two tubes) with Pem-mutE_F & Pem-mutE_R<br><i>+ E. coli transformations</i><br><br><b>PCR-purification:</b><br>257. pSB1C3-pemp (two tubes) with Pem-mutE_F & Pem-mutE_R<br>288. pSB1C3-pem (two tubes) with Pem-mutE_F & Pem-mutE_R<br><br><b>DpnI:</b><br>257. pSB1C3-pemp (two tubes) with Pem-mutE_F & Pem-mutE_R<br>288. pSB1C3-pem (two tubes) with Pem-mutE_F & Pem-mutE_R<br><br><h2>Results:</h2><br><b>Transformations:</b><br>All transformations spread on Bleomycin/zeocin have grown! If they are the right lactobacilli with the right plasmid remains to be seen, there seem to be a lot of contamination in the form of “clouds” covering all plates. Yet still there are larger colonies that very well could be what we want. Maybe the antibiotics are not efficient enough, we will try to spread normal plasmid free reuteri from frozen stock on the plates to investigate… We’ll also try a colony screening today with VF2 and VR. <br>Nothing grew on chloramphenicol plates, not even the positive control pVS2, though it grew on erythromycin both when transformed into reuteri and plantarum. <br><br><b>Gel electrophoresis:</b><br>All Pem/Pemp mutagenesises showed bands at the right lengths (~2700 bp), although they were a bit smudgy - weakly stretching backward toward the wells. The digests of Pem simply showed one band, a weaker counterpart of the uncut PCR-product. We suspect the digest didn’t go as supposed, maybe due to the PCR buffer. <br>The lactobacillus colony screenings showed up empty, perhaps the cells didn’t lyse? Or didn’t contain plasmids with VF and VR… We will continue to investigate the resistance problems, and await the sequencing results of the insertsite.<br>The screenings of e. coli transformations done thursday also gave us an empty gel, without even ladders. Possibly the gel had to shallow wells.</div>' | |
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Revision as of 22:05, 1 October 2013
