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Internal Restriction Sites

If any of the restriction enzymes you want to use are present in your gene, you need to remove them. For Biobrick 2.0 format, that would be BamHI, BglII, EcoRI, and XhoI. This section of the tutorial will explain one of many ways to achieve this.

As an example of this, let's look at a salicylate promoter basic part, Bca1111. This part confers exogenous salicylate-dependent transcription of downstream genes. Let me point out an unusual aspect of Biobrick basic parts before we dive into the construction file. A Biobrick basic part is defined as a sequence flanked by Biobrick restriction enzymes that has not been constructed based on biobrick standard assembly. In other words, anything that cannot be described as a composite part is a basic part. In the case of basic parts such as the ceaB open reading frame described in the first section, the open reading frame for the protein cannot be trivial deconstructed into a series of simpler sequences. Let's call this type of element a fundamental part. Therefore the part you designed is a "basic part" and a "fundamental part"--it can't be made any more basic. Biobrick part Bca1111, in contrast, contains several of these fundamental parts. It has an entire gene cassette of a promoter, ribosome binding site, the nahR open reading frame, and terminator. Together these parts produce the transcription factor protein, NahR. Additionally, Bca1111 contains the Psal promoter, which is activated by NahR. We could Biobrick all these fundamental parts, and then assemble something with the same utility as Bca1111 as a composite part. This procedure is the essence of refactoring--splitting a naturally-occuring sequence into its fundamental basic parts and reassembling them into a composite part that maintains the activity of the original. In some instances, this might be useful as subtle properties of the original cassette could be altered or improved by refactoring. In other instances, it just creates more work for you.

Alright, let's look at the construction file:

 Construction of salicylate promoter basic part
 PCR ca1110F/ca1111R on pBACr899     (814 bp, gp = A)
 PCR ca1111F/ca899R on pBACr899      (497 bp, gp = B)
 PCR ca1110F/ca899R on A+B           (1287 bp, EcoRI/BamHI)
 Digest pBca1100                     (EcoRI/BamHI, 2927+28, L)
 Product is pBca1100-Bca1111  {nahR-Psal}
 ca1110F  Forward EcoRI for Biobrick extreme variant of nahR-Psal  ctctggaattcatgAGATCTGCGATCCCGCGAAGAACC
 ca1111F  Removing the BglII site in nahR  catgaagtagatTtcgccaatgtc
 ca1111R  Removing the BglII site in nahR  gacattggcgaAatctacttcatg
 ca899R   Reverse BamHI for nahR promoter  GCAAAggatccTCTATGGTACTCGTGATGGC

Go ahead and download the 3 relavenent sequence files:

 File:JCAseq pBACr899.str
File:JCAseq pBca1100.str
File:JCAseq pBca1100-Bca1111.str

Open up pBACr899 in your editor. Predict what the product of PCR with oligos ca1110F and ca899R would be. Look for EcoRI/BamHI/BglII/XhoI restriction sites in that PCR product. Notice anything wrong? There is a single BglII site in the sequence. It must be removed, or the future use of BglII during assembly would result in internal cleavage of the part. This construction file will result in a basic part without the internal BglII site.

The construction file is telling you to perform 2 separate PCR reactions with the ca### oligos using pBACr899 as template. The names of those PCR products are "A" and "B". Oligos ca1111F and ca1111R will not match the template exactly, so you'll need to use the tricks described in the first tuturial to figure out where they would anneal. Go ahead and predict the products of those reactions. Now let's examine them.

Copy the last 24 bases of "A" and search for this sequence in "B". You should find it on the 5' (left) end of the sequence. This is the homology region between the two PCR products. Instead of using restriction enzymes on these PCR products, the construction file has you gel-purify, or gp them. This procedure will physically separate your shorter PCR products from the plasmid DNA template still present in the reaction.

In the next step, you set up another PCR reaction using a mixture of the gel-purified A and B fragments. The two oligos in the reaction anneal to the ends of the fragments. Notice that these oligos are the same two oligos you used in the first PCR simulation of this tutorial. They amplify the entire nahR-Psal cassette. Indeed, if you used pBACr899 as template for this reaction, you would obtain a PCR product that retained the internal BglII site. This is the reason we must separate out the template for the A and B pcrs prior to this third reaction, the assembly reaction.

The assembly reaction is an example of a non-canonical PCR reaction. Some people call it "SOEing," some call it "overlap PCR", and it is also somewhat similar to a method called "Quikchange". Here's what happens during the reaction:


As illustrated above, during the initial denaturation step of the PCR, everything becomes denatured into single strands. Upon annealing, the stands all anneal to homologous sequences. There are many potential ligation products. However, the only relavent ones are those that are substrates for polymerization. Do you recall the rules defining whether a double-stranded complex is sufficient to initiate polymerization? I've shown 4 of these products, and I leave it to you to figure out if there are additional products. Upon polymerization, only one of the annealing products can result in a full-length double stranded product containing the mutations present in the 24 bp annealing region. This product then becomes the substrate for PCR amplification with the oligos.

The rest of the construction file should look familiar to you from your previous tutorial exercises. Go ahead and simulate the rest of it and confirm that this results in the desired product.

Before we move on to the design section, let's take an aside and look at plasmid pBca1100. This plasmid is fairly similar to pBca9145. In fact, it matches pBca9145 exactly external to the EcoRI and XhoI sites. The difference is that a cassette is inserted between BamHI and XhoI containing a ribosome binding site, the red fluorescent protein (mRFP1) open reading frame, and a terminator. Without a promoter, pBca1100 doesn't confer production of the red protein product. Upon insertion of a transcription-initiating element within the BglII/BamHI region, the downstream gene should be expressed. I call pBca1100 an "RFP reporter" for Biobrick promoter basic parts. What do you suppose the phenotype is of cells harboring this plasmid upon growth in the presence or absence of exogenous salicylate? Think about it. This and other plasmids containing cassettes between EcoRI/BglII or between BamHI/XhoI can be useful during assembly of the Biobricks because they confer readily-observable phenotypes to the bacteria. Because these plasmids maintain the uniqueness of all 4 Biobrick enzymes, their relative positioning, and the specific locations of BamHI and BglII, they do not interfere with any (currently described) methods of Biobrick assembly.