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
PY54_RepA part sbb1141
PY54_RepA exists but needs PCR
This feature is a replication factor required for replication of a special origin of replication. For plasmids with that origin of replication, the plasmid will only replicate if the RepA protein is provided in trans. The protein is originally described in PMID: 15866931 and comes from a Yersinia bacteriaphage. Unfortunately, it is somewhat toxic to E. coli, and we want to know why and hopefully alleviate the problem. Though the sequence of this feature already exists, it does not exist as the rbs.cds part we need for our experiments. You can use the plasmid pBca9523-jtk2545 as the template in your construction file. In the end, it needs to be an rbs.cds! style part 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.
This part requires pBca1766
You have a toxic gene part that needs to be composed with pBca1766. This vector has the Pbad promoter upstream of the EcoRI site and the TrrnB terminator downstream of the BamHI site. So, you can clone your part as a basic part with the normal EcoRI/BamHI procedures and in the end get a plasmid that already has the needed regulation for our experiments. You should design your construction file to employ pBca1766-Bca1089.
P_cspB part sbb1116
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