pKGs2 is created to have several features not commonly found in cloning vectors. In order to make it easier to clone and work with DNA, I have included-
- Biobrick compatibility
- GoldenGate compatibility
- Ligase chain reaction compatibility
- Seamless-homology cloning (gibson, SLiCE)
- Modular backbone
- Negative selective marker
- M13F and M13R sequencing primers
- Transcriptional isolation
The Biobrick standard, or Biobrick RFC, is a popular standard because of its association with iGem. Though oftentimes cumbersome and time consuming to use, an extremely large amount of standard parts use this assembly. iGem’s website gives a good overview of this method.
However, I have purposely altered the standard in a few ways in order to make it better suited for compatibility with other methods.
Above is RFC, below is pKG. The prefixes of both are the same except for 1 modification, which is removal of the 1bp gap between NotI and XbaI because it is deemed unnecessary. The suffix differs more, as NotI is removed and an SbfI site is made by the addition of 2 nucleotides. This is done so both the prefix and suffix have an 8bp cutter as the outer restriction enzyme site. A homing endonuclease site is not additionally added because I-SceI is often used in selection schemes and other homing endonucleases are inefficient. Having 2 8bp cutters allows for DNAs assembled using alternative methods to be more open to additional biobrick cloning steps, as NotI and SbfI are far rarer than EcoRI and PstI. If required, the entire cassette can be replaced with a normal Biobrick cassette by cutting with EcoRI and PstI.
GoldenGate assembly is an assembly method based off of using Type IIS restriction enzymes. It allows for high efficiency cloning of DNAs in a modular fashion. Because it is still restriction enzyme based, sites need to be taken out of parts that will be assembled. However, parts can be put into plasmids for modular assembly, an advantage over gibson assembly and related techniques. Unlike BioBrick assembly, many parts can be put together at once.
Usual enzymes include BsaI and BsmBI. Below is an example of a 5 part 1 vector assembly.
Popular methods for GoldenGate include MoClo and GoldenBraid. Since these methods normally require many premade vectors, the base vector only supports insertion between 2 BsmBI sites derived from the base vector from the Yeast Toolkit. Parts can also be converted into GoldenGate compatible parts using SLiCE. Since part definitions were defined to be compatible with MoClo kits, MoClo base parts can be used in conjugation with normal parts for some assemblies.
Addgene gives a good overview of exactly how GoldenGate works.
BsaI- Use BsaI rather than BsaI-HF. BsaI apparently has better function with the T4 buffer at a higher temperature
BsmBI- More expensive than BsaI, also works at a higher temperature. Possibly better in the final cut reaction(?)
BbsI- Apparently needs storage at -80. Cuts 2bp away from recognition site instead of 1bp like BsaI or BsmBI
BtgZI- Cuts 10bp away from recognition site. Can be used to jump over BsaI. Extremely common in E coli genome
AarI/ (BfuAI, BspMI) – AarI is a 7bp cutter and includes BfuAI and BspMI. AarI is also extremely expensive, so avoiding this enzyme in modular cloning reactions is advisable
SapI – 7bp cutter. Cuts for overhang of only 3bp. Used in the Electra cloning system
The ligase cycling reaction, or LCR, was a protocol developed for in vitro DNA synthesis, but was adapted for in vitro DNA recombination. The ‘landmark’ paper goes into deep optimization of the procedure. Their method is rather complicated, and so there is a simpler, albeit less efficient, method as well.
LCR combines DNA in a modular fashion using overlapping primer(s) and phosphorylated PCR product(s). A simplified view is shown below.
Primer sequences called SOCs (Scaffold Oligonucleotide Connectors) are designed to have 20bp of overlap to both DNA ends which are desired to be connected. The user then adds the DNA and SOCs together and runs a thermocycling reaction. All the DNA deanneals. Once temperatures are lowered, the primers bind both ends and bring them together. Taq ligase then ligates the 2 fragments together.
This reaction is advantageous to gibson in that it has high efficiency for a larger number of fragments￼. In addition, the method is modular, with only new SOCs having to be bought for each new assembly. However, there are some drawbacks. As size increases, efficiency of the LCR reaction decreases. LCR also always requires a PCR reaction, and for simple 1 part cloning, takes 2 more primers than does SLiCE. It also can not effectively be done in a modular fashion because there are no type IIS restriction enzymes that are blunt cutters and have an over 6bp recognition sequence.
KG_Bricks offer compatibility with LCR with all base vectors having EcoRV sites between the Type IIS defined sites and BioBrick prefix and suffix ends. EcoRV can be used to create large amounts of vector with blunt phosphoylated ends, perfect for LCR cloning.