Difference between revisions of "Part:BBa K812000"

Latest revision as of 00:33, 27 October 2012

Biobricked pCS2+ plasmid for frogs and chicken

This plasmid is a biobricked version of the Xenopus standard plasmid pCS2+. This plasmid is made of a bacterial ampicilin resistant backbone, with a universal CMV promoter. The Biobrick site is surrounded by a a 5'UTR expression enhancer, and a ploy-A tail with a SV40 3' UTR that tnhance the stabilisation of the mRNA and the expression of the protein. pCS2+ can be used as expression vector in a wide variety of mammalian and avian cells. It is also functional in zebrafish embryos.

This plasmid is ment to be micro-injected in frog eggs or chicken eggs for a transient expression of the protein at the early ages of development of the embryo. This plasmid does not replicate inside the cells, so it gets diluted and degraded as the embryo develop and its cells multiply. And as long as there are no sequence homologies with the host genome, integration through recombination is null, leaving no chance for DNA propagation to the breeding.

To use this plasmid, you simply have to insert your gene between the biobrick prefix and suffix sites. It is better to add a kozac sequence in front of your coding sequences in order to enhance its expression.

This plasmid contains the standard registry insert J04450 as a negative cloning control.

This plasmid was characterized with different reporters.
For example, the insert J04450 is replaced by the BioBrick BBa_K812031 (sfGFP. This new plasmid (BBa_K812133) express sfGFP into Xenopus:
perdu

The expression of sfGFP is present only in one out of the two embryo/tadpole. sfGFP is present only in few tissue as others reporters. sfGFP is expressed in tail's muscles and branchial basket for this tadpole. After injection of this plasmid into Xenopus it does not diffuse in the eggs, and the plasmid express sfGFP in few different tissues.

In this picture, we see tadpoles that we injected with a solution of RNA coding for GFP. We injected 2.3nL of purified RNA at the concentration 100 ng/uL. The RNA was produced with the sp6 primer present in the pCS2+ backbone. perdu

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 4092
Illegal SpeI site found at 2
Illegal PstI site found at 16
Illegal NotI site found at 9
Illegal NotI site found at 248
Illegal NotI site found at 4098
21
INCOMPATIBLE WITH RFC[21]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 4092
Illegal BamHI site found at 4077
23
INCOMPATIBLE WITH RFC[23]
Illegal prefix found at 4092
Illegal suffix found at 2
25
INCOMPATIBLE WITH RFC[25]
Illegal prefix found at 4092
Plasmid lacks a suffix.
Illegal XbaI site found at 4107
Illegal SpeI site found at 2
Illegal PstI site found at 16
Illegal NgoMIV site found at 2611
1000
INCOMPATIBLE WITH RFC[1000]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal BsaI site found at 3532
Illegal BsaI.rc site found at 1634
Illegal SapI site found at 551

@@ Line 15: / Line 15: @@
 	<p>The expression of sfGFP is present only in one out of the two embryo/tadpole. sfGFP is present only in few tissue as others reporters. sfGFP is expressed in tail's muscles and branchial basket for this tadpole. After injection of this plasmid into <i>Xenopus</i> it does not diffuse in the eggs, and the plasmid express sfGFP in few different tissues.<br/>
 <br>
-In this picture, we see tadpoles that we injected with a solution of RNA coding for GFP. We injected 2.3nL of purified RNA at the concentration 100 ng/uL.
+In this picture, we see tadpoles that we injected with a solution of RNA coding for GFP. We injected 2.3nL of purified RNA at the concentration 100 ng/uL. The RNA was produced with the sp6 primer present in the pCS2+ backbone.
 <img src="https://static.igem.org/mediawiki/parts/c/cd/PCS2%2B_GFPaid_t0_%2B_IAA_%28c1%2Bc2%29.JPG" alt="perdu" width="880px" /> <br/><br/>
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