Difference between revisions of "Part:BBa K929001"

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

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

@@ Line 1: / Line 1: @@
 __NOTOC__
 <partinfo>BBa_K929001 short</partinfo>
+<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"
-! colspan="2" style="background:#66bbff;"|[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]]
 |-
 |'''BioBrick Nr.'''
-|[https://parts.igem.org/Part:BBa_929001 BBa_929001]
+|[https://parts.igem.org/Part:BBa_K929001 BBa_K929001]
 |-
 |'''RFC standard'''
-|[https://parts.igem.org/Help:Assembly_standard_10 RFC 10]
+|[https://parts.igem.org/Help:Assembly_standard_10 RFC 10] with aditional AgeI restriction site
 |-
 |'''Requirement'''
@@ Line 17: / Line 17: @@
 |-
 |'''Source'''
-|existing part: BBa_K103001
+|existing part: [https://parts.igem.org/Part:BBa_K103001 BBa_K103001]
 |-
 |'''Submitted by'''
 |[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>
-AID - Activation-induced cytidine deaminase
+[[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>
-The BioBrick modified AID is an improved version of the existing AID BioBrick (BBa_K103001). It has no NES(Nuclear Export Sequence) sequence and has NLS (Nuclear Localization Sequence) sequence and Kozak sequence added.
+'''AID - Activation-induced cytidine deaminase'''
+The BioBrick modified AID([https://parts.igem.org/Part:BBa_K929001 BBa_K929001]) is an improved version of the existing AID BioBrick ([https://parts.igem.org/Part:BBa_K103001 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:'''
 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:
+'''Functional NLS sequence:'''
 This part of the BioBrick directs the expressed protein into the nucleus, where it can mutate stronger.
-Kozak sequence
+'''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.<br> <br>
+<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