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James Macaulay



Welcome to your project page!

I've given you several parts to make.

  • Design oligos to make your part
  • Write up a proper construction file
  • Enter your Features, Oligos, Parts, and Plasmids into Clotho

You should design your construction strategy to put your part into plasmid vectorName-Bca1144 (Where vectorName is indicated for each part) using EcoRI and BamHI. The sequences for all plasmids involved in our project are available on Clotho.

It is essential that you make your part in the correct vector, so make sure you're using the right one for each part!

Several references have been provided to give you some background on the biology of your part.

Also, use the link above left to upload a picture of yourself, your name, and anything else you'd like, and rename the link from "Your Name Here" to your name.

Finally, you should create a notebook on the main page of the wiki

 lambda red/recA  part jtk2979

Lambda red/recA exists but needs to be moved

The lambda red genes encode proteins that enable efficient homologous recombination in E. coli. Many publications have described its use in genome manipulation experiments, and one of the more useful ones is PMID 10829079. Unfortunately, it is somewhat toxic to E. coli, and we want to know why and hopefully alleviate the problem. A composite part encoding all 4 CDS in one operon with ribosome binding sites and terminator but no promoter as part jtk2979. However, it is associated with the wrong vector (plasmid pBca9145-jtk2979). We need to move it into a new plasmid with vector pBca1766.

This part encodes a Toxic Gene

This part contains a slightly toxic gene. All our parts encoding toxic genes ultimately need to be Pbad.rbs.gene composite parts in a pUC/ColE1 plasmid. Follow the instructions for figuring out how to make this composite part. In some cases, the part already exists and you'll just need to move it into the right vector. Other ones will require some oligos and PCR. Others still just involve making composite parts.

 P_hdeAB part sbb1128

This part encodes a stress promoter

You will be cloning this stress promoter from E. coli MG1655 genomic DNA. You will find that your Feature has already been entered into Clotho, and you should design a Part from this Feature and name it with whatever sbb11## is indicated next to the Feature. Note that some of these stress promoters contain internal restriction sites. You'll need to take care of those as you did in the tutorials. Since these are promoters, not coding sequences, there is no easy way of predicting whether the mutations you introduce will be neutral or not. So, just pick a single point mutation to introduce to get rid of the restriction site. The vector for your plasmid should be pBjh1601KC. You should transform your ligation into Lefty cells