Difference between revisions of "Part:BBa K929000"
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| − | ! colspan="2" style="background: | + | ! colspan="2" style="background:rgb(240, 20, 70);"| CMV wtAID pA |
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! colspan="2"|[[Image:UP12_PlasmidCMV_wtAID_pA.png|300px]] | ! colspan="2"|[[Image:UP12_PlasmidCMV_wtAID_pA.png|300px]] | ||
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|[http://2012.igem.org/Team:Potsdam_Bioware Potsdam_Bioware2012] | |[http://2012.igem.org/Team:Potsdam_Bioware Potsdam_Bioware2012] | ||
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| − | The BioBrick "AID with CMV promoter and hGH-polyadenylation signal sequence" ([https://parts.igem.org/Part:BBa_K929000 BBa_K929000]) is an extended version of the existing AID BioBrick ([https://parts.igem.org/Part:BBa_K103001 BBa_K103001]). It is built of 3 parts: CMV promoter ([https://parts.igem.org/Part:BBa_I712004 BBa_I712004]), AID [https://parts.igem.org/Part:BBa_K103001 BBa_K103001])and hGH polyadenylation signal sequence ([https://parts.igem.org/Part:BBa_K404108 BBa_K404108]).<br> | + | [[Image:UP12_PlasmidCMV_wtAID_pA2.png|left|thumb|400px|Fig. 1: AID with CMV promoter and hGH-polyadenylation signal 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> |
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| + | The BioBrick "AID with CMV promoter and hGH-polyadenylation signal sequence" ([https://parts.igem.org/Part:BBa_K929000 BBa_K929000]) is an extended version of the existing AID BioBrick ([https://parts.igem.org/Part:BBa_K103001 BBa_K103001]). It is built of 3 parts: CMV promoter ([https://parts.igem.org/Part:BBa_I712004 BBa_I712004]), AID ([https://parts.igem.org/Part:BBa_K103001 BBa_K103001])and hGH polyadenylation signal sequence ([https://parts.igem.org/Part:BBa_K404108 BBa_K404108]).<br> | ||
'''AID''':<br> | '''AID''':<br> | ||
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. <br> | 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. <br> | ||
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CMV is an immediate-early Cytomegalovirus promoter for high-level expression. The CMV promoter is commonly used due to its very strong activity and effectivity in a broad range of cell types. The BioBrick is therefore improved via addition of the strong promoter. <br> | CMV is an immediate-early Cytomegalovirus promoter for high-level expression. The CMV promoter is commonly used due to its very strong activity and effectivity in a broad range of cell types. The BioBrick is therefore improved via addition of the strong promoter. <br> | ||
'''hGH polyadenylation signal sequence:'''<br> | '''hGH polyadenylation signal sequence:'''<br> | ||
| − | Polyadenylation is a significant part for the translation and stability of mRNA. In eukaryotes, it is part of the process that produces mature messenger RNA (mRNA) for translation. It, therefore, forms part of the larger process of gene expression. hGH terminator gives a signal to start polyadenylation in the translation process. | + | Polyadenylation is a significant part for the translation and stability of mRNA. In eukaryotes, it is part of the process that produces mature messenger RNA (mRNA) for translation. It, therefore, forms part of the larger process of gene expression. hGH terminator gives a signal to start polyadenylation in the translation process.<br> |
| − | + | <p style="background-color: rgb(238, 221, 130); font-weight: bold;">Characterization</p> | |
| + | '''Mutation rate''' in CHO-cells:<br> | ||
| + | [[Image:UP_12_mutation_rate.png|right|400px|thumb|Fig. 2: Comparison of the mutation rates between the wildtype AID, modified AID and modified AID-eGFP]] | ||
| + | We checked the mutation rate of wildtype AID([https://parts.igem.org/part:BBa_K929000 BBa_K929000]), modified AID([https://parts.igem.org/part:BBa_K929002 BBa_K929002]) and modified AID+eGFP([https://parts.igem.org/part:BBa_K929003 BBa_K929003]) (all expressed with CMV-promoter and hGHpolyA). Therefor we cotransfected CHO cells with a single chain construct and one of the AID versions. After certain expression time we purified the the single chain plasmids and transformed them into E.coli to enrich the mutated plasmids. After overnight culture and purification of the transformed plasmids, samples where sequenced. <br> | ||
| + | There is a significant higher mutation rate when wildtype AID, modified AID or modified AID-eGFP is added, compared to the same experiment without AID. Surprisingly, the mutation rate of wt AID is two times higher than the mutation rate of the modified AID or modified AID-eGFP (Fig. 2). This observation is contrary to our expectation. The modified AID+eGFP is located in the nucleus (Fig. 3), that means our construct works and should have a higher mutation rate. One possible explanation is that the modified AID has a very high mutation rate and therefore the transfected cells die or inactivate the plasmid like it was observed for the wildtype AID (Martin and Scharff (2002)).<br><br><br> | ||
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Latest revision as of 22:40, 29 September 2012
AID with CMV promoter and hGH-polyadenylation signal sequence
General information
| CMV wtAID pA | |
|---|---|
| |
| BioBrick Nr. | BBa_K929000 |
| RFC standard | RFC 10 |
| Requirement | pSB1C3 |
| Source | existing parts:(BBa_K103001;BBa_I712004; BBa_K404108) |
| Submitted by | [http://2012.igem.org/Team:Potsdam_Bioware Potsdam_Bioware2012] |
The BioBrick "AID with CMV promoter and hGH-polyadenylation signal sequence" (BBa_K929000) is an extended version of the existing AID BioBrick (BBa_K103001). It is built of 3 parts: CMV promoter (BBa_I712004), AID (BBa_K103001)and hGH polyadenylation signal sequence (BBa_K404108).
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.
CMV promoter:
CMV is an immediate-early Cytomegalovirus promoter for high-level expression. The CMV promoter is commonly used due to its very strong activity and effectivity in a broad range of cell types. The BioBrick is therefore improved via addition of the strong promoter.
hGH polyadenylation signal sequence:
Polyadenylation is a significant part for the translation and stability of mRNA. In eukaryotes, it is part of the process that produces mature messenger RNA (mRNA) for translation. It, therefore, forms part of the larger process of gene expression. hGH terminator gives a signal to start polyadenylation in the translation process.
Characterization
Mutation rate in CHO-cells:
We checked the mutation rate of wildtype AID(BBa_K929000), modified AID(BBa_K929002) and modified AID+eGFP(BBa_K929003) (all expressed with CMV-promoter and hGHpolyA). Therefor we cotransfected CHO cells with a single chain construct and one of the AID versions. After certain expression time we purified the the single chain plasmids and transformed them into E.coli to enrich the mutated plasmids. After overnight culture and purification of the transformed plasmids, samples where sequenced.
There is a significant higher mutation rate when wildtype AID, modified AID or modified AID-eGFP is added, compared to the same experiment without AID. Surprisingly, the mutation rate of wt AID is two times higher than the mutation rate of the modified AID or modified AID-eGFP (Fig. 2). This observation is contrary to our expectation. The modified AID+eGFP is located in the nucleus (Fig. 3), that means our construct works and should have a higher mutation rate. One possible explanation is that the modified AID has a very high mutation rate and therefore the transfected cells die or inactivate the plasmid like it was observed for the wildtype AID (Martin and Scharff (2002)).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1661
Illegal BsaI.rc site found at 740
Illegal SapI site found at 841