From 2011.igem.org
(Difference between revisions)
|
|
| (9 intermediate revisions not shown) |
| Line 1: |
Line 1: |
| - | =Introduction=
| |
| | | | |
| - | The Ribozyme Project aims to establish a novel way to make fusion proteins, therefore, it is important to ensure that the intron components will effectively remove an interrupting sequence and effectively re-ligate together, ensuring a functional protein product. Consequently, an easily detectable protein product must be interrupted with a sequence that is definite to either stop translation or alter the protein enough to render it ineffective. In our design, GFP is interrupted with a stop codon-containing sequence, which is flanked by the two intron parts. The parts submitted for this design were sequence and subsequently ligated into PSB1C3.
| |
| - |
| |
| - |
| |
| - | =Making the Construct with <nowiki>RFC53</nowiki>=
| |
| - |
| |
| - | [[File:rfc53overview.png|none|thumb|500px|'''Figure 6''': Making the Construct]]
| |
| - |
| |
| - | '''Figure 6''' is a flow chart of the general work-flow involved in the construction of our experimental plasmid, as per <nowiki>RFC53</nowiki> conventions. The enzymes used are SacI, EarI and SapI, however, Bg1II could have been used if ligation were done in the opposite direction.
| |
| - |
| |
| - | *'''(1)''' The insert is isolated through a series of enzyme digestions. As shown, the plasmid is initially cut by SacI. One Intron (in blue) is shown here as a representation. Note that SacI is the first enzyme to cut in the meta-prefix, instead of EarI because of the TCGA complementary overhand needed for ligation in '''(3)'''. Next, EarI cuts at the meta-suffix to produce the CCA overhang also necessary for the step '''(3)''' ligation. The desired intert (containing the intron) is isolated for subsequent use.
| |
| - |
| |
| - | *'''(2)''' Similarly, the PSB1C3 vector is isolated through enzyme digestion. Note that "N" indicates that this is the vector portion. SapI cuts in the meta-prefix region to produce the GGT overhang (complimentary to CCA in '''(1)'''). Next, SacI further cuts in the meta-prefix to produce the other sticky end (AGCT). The vector is also isolated for the ligation step.
| |
| - |
| |
| - | *'''(3)''' The two components (insert and vector) are ligated together to produce the final construct.
| |
| - |
| |
| - | *'''(4)''' According to the experimental design, the final construct will contain self-excising ribozymes, which in the last step result in a non-disruptive ligation scar and, therefore, the expression of GFP.
| |
Latest revision as of 16:52, 23 September 2011
"
