iGEM Meeting #4 07/16/08 Presentation by Robert Ovadia Lactose Intolerance iGEM Meeting #4 07/16/08 Presentation by Robert Ovadia I chose this background because it looks like it had two plasmids. It relates to my project because to solve lactose intolerance, we need two plasmids. The first being *click*

iGEM Meeting #4 07/16/08 Presentation by Robert Ovadia System Design The idea behind using RBS’s is we want to use the weakest RBS for lacY so we can pump the strongest promoter. We want to express LacZ as much in order to do this. *ADD ORI’s* iGEM Meeting #4 07/16/08 Presentation by Robert Ovadia

iGEM Meeting #4 07/16/08 Presentation by Robert Ovadia System Design By next week, you guys should be telling me how the plasmids look like. I will test you! *ORI’s* iGEM Meeting #4 07/16/08 Presentation by Robert Ovadia

Last Week’s Results What we had We have sequenced: We have transformed: LacY 34 LacZ Into pSB1A2 LacY Purified ready to cut: From PCR Lysis Cassette We will send another one in for sequencing to determine what really happened. There was a single base mutation. It would have been nice if it didn’t change the amino acid, but unfortunately it did. We are hesitant to use this construct because 1 mutation could alter the folding of the protein and the function. So all we have now is LacY in pSB1A2 *MAKE SIMPLER* PCR Purified 31 LacY 31 33 33 LacY iGEM Meeting #4 07/16/08 Presentation by Robert Ovadia

Last Week’s Results Sequencing: The Work Chart Did it work?  31 LacY  33 LacY  34 LacZ We will send another one in for sequencing to determine what really happened. There was a single base mutation. It would have been nice if it didn’t change the amino acid, but unfortunately it did. We are hesitant to use this construct because 1 mutation could alter the folding of the protein and the function. So all we have now is LacY in pSB1A2 *MAKE SIMPLER*  Lysis Cassette iGEM Meeting #4 07/16/08 Presentation by Robert Ovadia

Future Goals What’s next? 34 LacZ In J23100 Str. Po. Lysis Cassette In pSB1A2 In pSB2K3 31 LacY We have our three constructs that we currently have. Now what do we want to do this them for next week? LacY into 31/33, the weaker RBS’s 0.01 and 0.07 (7 fold increase). From 33 to 32, 4 fold increase. LacY + LacZ into TT Primer design for mutagensis Lysis cassette into pSB1AK3. Photo of lysis cassette. *ADD MORE* In pSB1AK3 33 LacY In pSB1AK3 iGEM Meeting #4 07/16/08 Presentation by Robert Ovadia

iGEM Meeting #4 07/16/08 Presentation by Robert Ovadia Current Constructs We have sequenced: Preparing to digest: A whole lot of stuff… Three promoters Lac. inducible promoters (A81, B4) RBS, GFP ,TT Our current constructs B0015 (1AK3 + 2K3) LacY 31 LacY 33 LacY *UPDATE* 34 LacZ Lysis Cassette iGEM Meeting #4 07/16/08 Presentation by Robert Ovadia