Template:SBB11Projectstdt1114

Justin Wang

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

 SssI methyltransferas  part jtk2259

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.

This part exists but requires an EcoRI/BamHI transfer

The SssI methyltransferase methylates DNA at sites analogous to CpG methylation in eukaryotes. It comes from a bacterium, and it is sold in recombinant form by NEB. Their website (http://www.neb.com/nebecomm/products/productM0226.asp) has more information and links about the protein. Unfortunately, it's a little toxic, and we want to see if we can figure out why and if we can optimize its expression through use of a stress promoter. This part already exists, but it is with the wrong vector. You'll need to do an Eco/Bam transfer to create the right plasmid. Your starting material is pBca9145-jtk2259, and we need to subclone it into pBca9523-Bca1144#5.

 P_fimF/I part sbb1110

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

 P_cysI part sbb1129

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