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Team:MoWestern Davidson/FSLs
From 2009.igem.org
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To encode the SAT problem into the reporter gene we needed to mutate the reporter genes that we wanted. To do so we employed PCR mutatgenesis. By using a forward primer that contains the biobrick prefix, ATG, our desired mutation, and then the 15nt after the first codon of the gene we were able to insert the mutations. We have several different constructs for determining how many clauses are satisfied. The first construct was to test the suppression ability of our tRNAs. For this we simply inserted a 5nt sequence that matches one of our tRNAs. | To encode the SAT problem into the reporter gene we needed to mutate the reporter genes that we wanted. To do so we employed PCR mutatgenesis. By using a forward primer that contains the biobrick prefix, ATG, our desired mutation, and then the 15nt after the first codon of the gene we were able to insert the mutations. We have several different constructs for determining how many clauses are satisfied. The first construct was to test the suppression ability of our tRNAs. For this we simply inserted a 5nt sequence that matches one of our tRNAs. | ||
<br /> | <br /> | ||
| - | <br />atg CCCUC gct …<br /><br /> | + | <br /><font face="courier new">atg CCCUC gct …</font><br /><br /> |
The second construct is one 2-SAT logical clause. In this case if one of the suppressor tRNAs that is encoded in the logical clause is present in the cell, then the frameshift will be suppressed and the reporter will be expressed. | The second construct is one 2-SAT logical clause. In this case if one of the suppressor tRNAs that is encoded in the logical clause is present in the cell, then the frameshift will be suppressed and the reporter will be expressed. | ||
| - | <br /><br />aug <span style="color:#ff0000">VVVVV g VVVVVV</span> gct ... | + | <br /><br />aug <span style="color:#ff0000"><font face="courier new">VVVVV g VVVVVV</span> gct ...</font> |
<br /><br /> | <br /><br /> | ||
| - | In the above example CCCUC g CCACC represents the logical clause (A or B). If neither 5base insertion is suppressed, then the translation will be off track when it gets to the gct (the 2nd codon in unmutated RFP). If either tRNA is present and suppresses its insertion, then the frame will be restored when the translation gets to gct. | + | In the above example <font face="courier new">CCCUC g CCACC</font> represents the logical clause (A or B). If neither 5base insertion is suppressed, then the translation will be off track when it gets to the gct (the 2nd codon in unmutated RFP). If either tRNA is present and suppresses its insertion, then the frame will be restored when the translation gets to gct. |
Revision as of 19:32, 28 July 2009
FSL Design:
Table:
LC1 : VVVVV g VVVVV
LC2 : XXXXX g XXXXX
LC3 : YYYYY g YYYYY
LC4 : ZZZZZ g ZZZZZ
To encode the SAT problem into the reporter gene we needed to mutate the reporter genes that we wanted. To do so we employed PCR mutatgenesis. By using a forward primer that contains the biobrick prefix, ATG, our desired mutation, and then the 15nt after the first codon of the gene we were able to insert the mutations. We have several different constructs for determining how many clauses are satisfied. The first construct was to test the suppression ability of our tRNAs. For this we simply inserted a 5nt sequence that matches one of our tRNAs.
atg CCCUC gct …
The second construct is one 2-SAT logical clause. In this case if one of the suppressor tRNAs that is encoded in the logical clause is present in the cell, then the frameshift will be suppressed and the reporter will be expressed.
aug VVVVV g VVVVVV</span> gct ...
In the above example CCCUC g CCACC represents the logical clause (A or B). If neither 5base insertion is suppressed, then the translation will be off track when it gets to the gct (the 2nd codon in unmutated RFP). If either tRNA is present and suppresses its insertion, then the frame will be restored when the translation gets to gct.
