Part:BBa_K4895995
Micro-Knock-Out-Plasmid(MiKOP)
This part is to serve as a template for designing and integrating you own micro-plasmid Knock Out.
Knock Out Plasmids
Often, teams may seek to knock out genomic expression of specific enzymes or regulatory sequences in bacteria, as their one, circularized chromosome is relatively simple to recombine with. While recombination events may be rare, it is often that this low statistical probability can be circumvented with higher efficiency transformations as well as higher quality transformants. However, in the past, recombination often requires a separate bacterium along with a full expression plasmid. For instance, it is commonplace to create a full plasmid that includes an origin of replication that may not be compatible with the targeted strain of bacteria, as a way of easily cloning the plasmid and repairing any nicks in a separate cloning step. It is understandable that using a different strain or even species of bacteria can be daunting, and generating or buying a full plasmid can be pricey. Along with this, the old method of recombination requires two recombination sites, each around 300bp long. Recombination events are rare enough, and adding on the need to generate two recombination events only makes things worse. Fortunately, ASU iGEM has the solution:
Introducing: MiKOP, or Micro Knock Out Plasmid
This year, we have inadvertently produced a novel technique that is both more accessible and relatively simple to design and build with. However, it must be stressed that this KO technique only applies to genes that are directly downstream of promoter regions, as well as genes that are not polycistronically expressed with vital genes. With that out of the way, let's begin. The design of an MiKOP is very simple, with only 4 major parts:
- 600+bp recombination site
- 2 constitutive terminators, upstream of the recombination site
- Antibiotic resistance, upstream of the two terminators
- Constitutive promoter, upstream of the antibiotic resistance, and facing away from the recombination site.
The rationale here is simple: Why use a separate organism for plasmid repair and cloning when it is possible to be done in vitro. The microplasmid takes a bare-bones approach to the KO gene problem. The 600bp recombination site is to increase the chances of recombination, as well as increase the specificity of the recombination site. The 2 constitutive terminators serve as the "KO" mechanism; because we are inserting directly between the gene and its promoter, we know that by inserting two terminators, it definitively shuts down gene expression. Finally, the antibiotic resistance and constitutive promoter serve as a selection mechanism; it is a way of directly confirming the success of the recombination without needing to sequence genomically en masse.
How to use this part
This part serves as a template for designing the KO plasmid itself. Please be extra careful and note the direction that everything is pointing. For instance, make sure that the promoter, antibiotic resistance, and two terminators are all in the same direction. Furthermore, make sure to deeply analyze the region that recombination may occur, that way you may avoid messy and/or shutting down other genes you haven't planned on knocking out.
Colony PCR Results
The following is an image from our colony PCR that confirmed the successful recombination of MiKOP
The left four DNA wells (not counting the ladder) represent colonies picked from a 1 hour outgrowth period, while the right four DNA wells represent colonies picked from a 3 hour outgrowth period. Due to pipetting error, the second well has less DNA in it compared to the others.
While the results were more or less the same, the 3 hour outgrowth plate demonstrated significantly more growth compared to the 1 hour outgrowth plate.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1273
Illegal NheI site found at 1449
Illegal NheI site found at 1472 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |