Difference between revisions of "Part:BBa K929001"
 
(6 intermediate revisions by 2 users not shown)
Line 3: Line 3:
 
<p style="background-color: rgb(238, 221, 130); font-weight: bold;">General information</p>
 
<p style="background-color: rgb(238, 221, 130); font-weight: bold;">General information</p>
 
{| style="color:black" cellpadding="6" cellspacing="1" border="2" align="right"
 
{| style="color:black" cellpadding="6" cellspacing="1" border="2" align="right"
−
! colspan="2" style="background:rgb(240, 20, 70);"|[https://parts.igem.org/Part:BBa_K929001 AID without NES, with NLS and Kozak sequence]
+
! colspan="2" style="background:rgb(240, 20, 70);"| AID without NES, with NLS and Kozak sequence
 
|-
 
|-
 
! colspan="2"|[[Image:UP12_PlasmidAID without NES, with NLS and Kozak sequence.png|300px]]
 
! colspan="2"|[[Image:UP12_PlasmidAID without NES, with NLS and Kozak sequence.png|300px]]
Line 22: Line 22:
 
|[http://2012.igem.org/Team:Potsdam_Bioware Potsdam_Bioware2012]
 
|[http://2012.igem.org/Team:Potsdam_Bioware Potsdam_Bioware2012]
 
|}
 
|}
−
<br>
+
 
−
[[Image:UP12_PlasmidAID without NES, with NLS and Kozak sequence2.png|left|thumb|400px]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>
+
[[Image:UP12_PlasmidAID without NES, with NLS and Kozak sequence2.png|left|thumb|400px|Fig. 1: AID without NES, with NLS and Kozak sequence in pSB1C3 vector]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>
  
 
'''AID - Activation-induced cytidine deaminase'''
 
'''AID - Activation-induced cytidine deaminase'''
Line 48: Line 48:
  
 
<p style="background-color: rgb(238, 221, 130); font-weight: bold;">characterization</p>
 
<p style="background-color: rgb(238, 221, 130); font-weight: bold;">characterization</p>
 +
 +
To characterize the "modified AID" we added a (strong) CMV promoter and a hGH polyadenylation sequence. See the characterization of the resulting part ([https://parts.igem.org/part:BBa_K929002 BBa_K929002]). To see the localization,transfection success and make FACS possible we further fused "modified AID" with eGFP.See the characterization of the resulting part([https://parts.igem.org/part:BBa_K929003 BBa_K929003]).
 +
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Latest revision as of 22:41, 29 September 2012

AID without NES, with NLS and Kozak sequence

General information

AID without NES, with NLS and Kozak sequence
UP12 PlasmidAID without NES, with NLS and Kozak sequence.png
BioBrick Nr. BBa_K929001
RFC standard RFC 10 with aditional AgeI restriction site
Requirement pSB1C3
Source existing part: BBa_K103001
Submitted by [http://2012.igem.org/Team:Potsdam_Bioware Potsdam_Bioware2012]
Fig. 1: AID without NES, with NLS and Kozak sequence in pSB1C3 vector





















AID - Activation-induced cytidine deaminase

The BioBrick modified AID(BBa_K929001) is an improved version of the existing AID BioBrick (BBa_K103001). It has no NES(Nuclear Export Sequence) sequence and an aditional NLS (Nuclear Localization Sequence) sequence and Kozak sequence added.

The AID motif is naturally terminated with the Nuclear Export Sequence (NES) that causes the protein to translocate from the nucleus to the cytoplasm. Additionally, upstream, dysfunctional Nuclear Localization Sequence (NLS) is located. Due to the fact that AID mutates the actively transcribed single stranded DNA, it is supposed that the direction of the enzyme to the inside of the nucleus would improve the mutation rate.

AID:

AID is known to be responsible for somatic hypermutation and the class-switch recombination of immunoglobulin in B cells. This enzyme of 28 kDa originally occurs in B cells but does also show activity after transfection into CHO cells. AID induces the deamination of cytidine to uridine at actively transcribed single strand DNA. The replacement of cytidine by uridine leads to a mismatch during DNA replication and integrates a single base substitution predominantly in the immunoglobulin genes.

Functional NLS sequence:

This part of the BioBrick directs the expressed protein into the nucleus, where it can mutate stronger.

Kozak sequence

Kozak consensus sequence is added upstream of the AID mutant to express the protein stronger.

Aditional AgeI restriciton site

An aditional AgeI restriciton site was added at the 3` end in front of the stop codon "taa". Therefore fusion with RFC 25 parts at the C-terminus of the modified AID is possible. Incompatibility with RFC 25 is displayed because we defined the AgeI restriction site to be in the insert. Actually because of this AgeI site, fusion with RFC 25 parts is possible.

characterization

To characterize the "modified AID" we added a (strong) CMV promoter and a hGH polyadenylation sequence. See the characterization of the resulting part (BBa_K929002). To see the localization,transfection success and make FACS possible we further fused "modified AID" with eGFP.See the characterization of the resulting part(BBa_K929003).

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 579
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 108
    Illegal SapI site found at 209