Difference between revisions of "Part:BBa K2077000"

Latest revision as of 00:50, 22 October 2016

pSB416-GPD: URA3-selectable S. cerevisiae Expression Vector (RFC 10)

This is a RFC10 standard yeast expression vector adapted from the widely used plasmid p416-GPD. The MCS of p416-GPD was replaced with the standard BioBrick prefix and suffix and an illegal PstI restriction site near the URA3 promoter of p416-GPD was removed. In the resultant plasmid, pSB416-GPD (5743 bp), the BioBrick prefix and suffix is situated between the strong constitutive yeast GPD promoter (TDH3) and CYC1 terminator allowing for facile cloning and expression of translational units in yeast. Standard VF2 and VR primer binding sites are included and M13 primer binding sites bracket the promoter and terminator.

The plasmid has a ColE1 origin and ampicillin resistance for replication in E. coli. It also contains a yeast CEN/ARS and URA3-selectable marker to provide high fidelity maintenance and low copy number in S. cerevisiae.

Figure 1. Top Left: Yeast with insertless pSB416-GPD under fluorescent light. Top Right: Yeast with insertless pSB416-GPD under normal light. Bottom Left: Yeast with insert under fluorescent light. Bottom Right: Yeast with insert under normal light

The promoter, terminator, biobrick prefix and suffix, and selectable markers in pSB416-GPD all function as expected. Post cloning mls-yeGFP into the vector, E.coli transformed with it grew on LB+Amp plates verifying the function of the plasmids amp resistance. Yeast transformed with the vector grew in CSM-Ura media regardless of mls-yeGFP's presence or absence in the vector, and yeast with mls-yeGFP fluoresce when excited with blue light, indicating that the Ura3 selectable marker, the GPD promoter, they CyC1 terminator, and the biobrick prefix and suffix function (Figure 1). 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 5722
Illegal SpeI site found at 2
Illegal PstI site found at 16
Illegal NotI site found at 9
Illegal NotI site found at 5728
21
INCOMPATIBLE WITH RFC[21]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 5722
23
INCOMPATIBLE WITH RFC[23]
Illegal prefix found at 5722
Illegal suffix found at 2
25
INCOMPATIBLE WITH RFC[25]
Illegal prefix found at 5722
Plasmid lacks a suffix.
Illegal XbaI site found at 5737
Illegal SpeI site found at 2
Illegal PstI site found at 16
Illegal NgoMIV site found at 603
1000
INCOMPATIBLE WITH RFC[1000]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal BsaI site found at 3703
Illegal BsaI.rc site found at 1195
Illegal SapI site found at 1347
Illegal SapI.rc site found at 2681
Illegal SapI.rc site found at 4785

@@ Line 7: / Line 7: @@
 The plasmid has a ColE1 origin and ampicillin resistance for replication in E. coli. It also contains a yeast CEN/ARS and URA3-selectable marker to provide high fidelity maintenance and low copy number in S. cerevisiae.
-<!-- Add more about the biology of this part here
+[[File:T--RHIT--FluorsenceComparisonpicture.PNG|200px|thumb|left|Figure 1. Top Left: Yeast with insertless pSB416-GPD under fluorescent light. Top Right: Yeast with insertless pSB416-GPD under normal light. Bottom Left: Yeast with insert under fluorescent light. Bottom Right: Yeast with insert under normal light]]
+<br>
+The promoter, terminator, biobrick prefix and suffix, and selectable markers in pSB416-GPD all function as expected. Post cloning mls-yeGFP into the vector, E.coli transformed with it grew on LB+Amp plates verifying the function of the plasmids amp resistance. Yeast transformed with the vector grew in CSM-Ura media regardless of mls-yeGFP's presence or absence in the vector, and yeast with mls-yeGFP fluoresce when excited with blue light, indicating that the Ura3 selectable marker, the GPD promoter, they CyC1 terminator, and the biobrick prefix and suffix function (Figure 1).<!-- Add more about the biology of this part here
 ===Usage and Biology===
-[[File:T--RHIT--FluorsenceComparisonpicture.PNG|200px|thumb|left|Figure 1. Top Left: Yeast under fluorescent light. Top Right Yeast under Normal light. Bottom Left: Yeast with insert under fluorescent light. Bottom Right: Yeast with insert under normal light]]
-The promoter, terminator, biobrick prefix and suffix, and selectable markers in pSB416-GPD all function as expected. Post cloning mls-yeGFP into the vector, E.coli transformed with it grew on LB+Amp plates verifying the function of the plasmids amp resistance. Yeast transformed with the vector grew in CSM-Ura media regardless of mls-yeGFP's presence or absence in the vector, and yeast with mls-yeGFP fluoresce when excited with blue light, indicating that the Ura3 selectable marker, the GPD promoter, they CyC1 terminator, and the biobrick prefix and suffix function (Figure 1).
 <!-- -->