Difference between revisions of "Team:HSHL/Design"
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<td> AhHMA3 cDNA</td> | <td> AhHMA3 cDNA</td> | ||
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<td> <span class="highlightGreen">highlighted</span></td> | <td> <span class="highlightGreen">highlighted</span></td> | ||
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<td><b>whole sequence</b> </td> | <td><b>whole sequence</b> </td> | ||
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<p> In the following the whole promoter and gene sequence including the restriction sites to ligate into the vector pSB1C3 is shown. | <p> In the following the whole promoter and gene sequence including the restriction sites to ligate into the vector pSB1C3 is shown. | ||
<br><span class="highlightDarkBlue">GA<span class="highlightRed">G</span>TTC</span> = changed codon to make sure that EcoRI will not cut several times</p> | <br><span class="highlightDarkBlue">GA<span class="highlightRed">G</span>TTC</span> = changed codon to make sure that EcoRI will not cut several times</p> | ||
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<td> Total length of the synthesized fragments</td> | <td> Total length of the synthesized fragments</td> | ||
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<td> Length of the sequence after the ligation</td> | <td> Length of the sequence after the ligation</td> | ||
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Latest revision as of 20:42, 17 October 2018
Design
1st idea
- Working with the whole sequence
The HMA3 Sequence is with 2619 bp quite big which make it a little bit difficult for us to actually isolate this sequence out of the A. halleri. That is the reason why we decided to get it synthesized by IDT.
First option was to use the whole sequence (2631 bp). A CaMV 35S (universal promoter, Cauliflower Mosaic Virus) is used as a promoter with a length of 345 bp.
For our Biobrick we decided to use the Vector called pSB1C3 with a chloramphenicol resistance. By including the sequence of the restriction sites of EcoRI at the beginning and PstI at the end of our sequence, which are the same restriction sites for the plasmide, we can insert our sequence into our targeted vector. In our specific case EcoRI would cut about two times into our Gene HMA3 for example. To make sure the restriction enzyme will not cut several times into our sequence we had to change some codons in the original sequence of our Gene HMA3 (you can see the changes of the codons below in red).
| Sequence | Length [bp] | Color |
|---|---|---|
| CaMV 35S promoter | 345 | letters |
| AhHMA3 cDNA | 2274 | highlighted |
| Restriction site of EcoRI (G/AATTC) | 6 | highlighted |
| restriction site of PstI (CTGCA/G) | 6 | highlighted |
| whole sequence | 2613 |
Promoter and gene sequence
In the following the whole promoter and gene sequence including the restriction sites to ligate into the vector pSB1C3 is shown.
GAGTTC = changed codon to make sure that EcoRI will not cut several times
2nd idea
- Separating the sequence
The second option is to separate our whole promoter and gene sequence into 4 fragment parts. You can see the parts in the table to the right-hand side.
| |
|||||
| Sequence | Length [bp] | beginning of the sequence | end of the sequence | ||
|---|---|---|---|---|---|
| IDT 1. Part sequence |
614 | EcorRI: G/AATTC | HindIII: A/AGCTT | ||
| IDT 2. Part sequence |
593 | HindIII: A/AGCTT | SmaI: CCC/GGG; blunt end | ||
| IDT 3. Part sequence |
556 | SmaI: CCC/GGG; blunt end | EcoRII: -/CCWGG; W=A/T | ||
| IDT 4. Part sequence |
885 | EcoRII: -/CCWGG; W=A/T | PstI: CTGCA/G | ||
| Total length of the synthesized fragments | 2648 | ||||
| Length of the sequence after the ligation | 2613 | ||||
The following part shows the 4 fragments, which were synthesized by the IDT. The black letters indicates the promoter sequence and the green letters the HMA3 gene sequence. The restriction sites for each restriction enzyme are highlighted in different colors to clearly differentiate inbetween them. To ligate these fragments into the vector pSB1C3, the first fragment needs to have the restriction site of EcoRI at the beginning as well as the fourth fragment needs to end with the restriction site of PstI. For this sequence design, we also have to changed a few codons to make sure that EcoRI and HindIII will not cut several times in the sequence. All four fragments will bind together because of the different restriction sites. For example, the end of the first fragment and the beginning of the second fragment have the same restriction site. This means only one end fits the other end.
1st fragment
Length: 614 bp
Restriction enzymes
EcoRI: G/AATTC
HindIII: A/AGCTT
GAGTTC = changed codon to make sure that EcoRI will not cut several times
2nd fragment
Length: 593 bp
Restriction enzymes
HindIII: A/AGCTT
SmaI: CCC/GGG; blunt end
3rd fragment
Length: 556 bp
Restriction enzymes
SmaI: CCC/GGG; blunt end
EcoRII: -/CCWGG; W=A/T
AAGCT = changed codon at the restriction site to make sure that HindIII will not cut several times
4th fragment
Length: 885 bp
Restriction enzymes
EcoRII: -/CCWGG; W=A/T
PstI: CTGCA/G
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