Question

Molecular Bio lab. HELP!!

In the files section for your group there is a simulated output from an automated DNA sequencer using a variation of the classic Sanger method. (If you want to print it, it is formatted for legal sized paper.) This sequence encodes a protein that you wish to study further by cloning the gene. The protein of interest has a molecular weight of at least 10 kDa. The sequence was sequenced by a primer walking approach, but unfortunately the correct order of the sequence traces was lost (really bad lab notebook skills). Your tasks: Determine the correct order of the sequence traces and the entire sequence. • Locate an open reading frame that could encode the protein of interest. Determine the amino acid sequence, the molecular weight, and the isoelectric point of this protein. Design a set of PCR primers that will amplify at a minimum this entire coding region. These primers must pass all the requirements for good primers. Develop a cloning strategy that would enable you to clone at a minimum the coding region from this sequence into the vector pUC19, so that the coding region is in a clockwise orientation (google "pUC19 sequence"). There are several ways to accomplish this cloning objective; the only requirement is that it is theoretically possible and that you can show that the coding region is in a clockwise orientation. You may assume that you have a reasonably competent lab technician to carry out the experimental work, so you don't need detailed instructions for each experiment, however they do need instructions as to what to look for at each step. For example: • It is sufficient to say digest the sequence with restriction enzyme BstEll, and run a gel. They know how to do that, but you would have to tell them what sizes of fragments to look for on the gel. (If that information is important to your strategy.) • You can say transform bacteria with this DNA, and they know how to do that, however you would need to tell them what selection to use and, if you are using a blue/white screening system what color of colony they should look for.

Here is the first part: the sequence traces and the entire sequence. i just need the last 3 tasks. i color coded the ends so you can see where it overlaps and connects

Answer 1

1) The correct order of the sequence traces =

Trace 2, trace 1, trace 5, trace 4 and then trace 3

2) The protein sequence is attached below- (via expasy) frame 2- open reading frame given below-

3) The MW= 12989.24 KDa

a N l F (64 A S F e - S R T C P H A S L A 7:43 AM O M G V tcc tcc acc S S T 5'3' Frame 2 a - 02 م G Qo K . gcag cct gtt agt cgg tct tac tgc tgc tgc ggt aag tog ggc gaa ttc gag ctc ggt acc Q P V S R S Y C C C G K S G E F E L G T cgggga tcctct atg ccc cga gcg gga ctc cgt ttt ttg goc gcg ttg acgggg tog gca R G S S M P R A G L R F L A A L T G S A ctt gct cgg gct gat tca ttg gaa atg cgc aaa caa ttg gca atg cag tca cgc gtt tgg L A R A D S L E M R K Q L A M Q S R V W gtt cgc ggc atc cgc acg tgt ctt cag aca cgt agg gat aca tta cag ggc gac tgt ctg V B G I B T C L Q T B B D T L C G D C L. ccg tgc aga ata cta tct ctc aag gtt gct gta ata cgc gag gca atg tgc tgg cag acg P C R I L S L K V A V I R E A M C W Q T acg tat agc cgt tta ccg tat gat cac gct tgc gaa a ac gcg agg tct ata ggt ctc gco T Y S R L P Y D H A C E N A R S I G L A cgc cca tgg cca acc aaa cct gtt tgc aaa cac ggt ttt gtt aca gac gaa gtc acc tga R P W Ρ Τ Κ Ρ V C Κ Η G F V Τ D E V T tgg gtg tat cgt ttc aat aga gtc gga cct gca ggc atg caa gct tgg cca act tog cgt W V Y R F N R V G P A G M D A W P T S R cct cca cct P P P. 5'3' Frame 3-

And isoelectric point is 9.88