Difference between revisions of "Part:BBa K4361214"
| Line 18: | Line 18: | ||
<ul> | <ul> | ||
<li>[[Part:BBa_K4361204]]: R4</li> | <li>[[Part:BBa_K4361204]]: R4</li> | ||
| − | <li>[[Part:BBa_K4361205]]: F4. | + | <li>[[Part:BBa_K4361205]]: F4.1 A40V</li> |
</ul> | </ul> | ||
</li> | </li> | ||
| Line 57: | Line 57: | ||
</ul> | </ul> | ||
| − | This primer contains a mutation that changes the codon for alanine (A, GCG) in position 62 to that of lysine (K, AAA). <br> | + | This primer contains a mutation that changes the codon for alanine (A, GCG) in position 62 to that of lysine (K, AAA).<br> |
<br> | <br> | ||
Revision as of 14:55, 14 September 2022
BlcR BTB primer F7.4 A62K
For the site-directed mutagenesis (SDM) of our protein of interest, BlcR, multiple sets of back-to-back (BTB) primers were designed. The 5' ends of these primers anneal to adjacent nucleotides such that the full plasmid containing the protein (Part:BBa_K4361106) can be replicated. Each of these numbered sets of primers contains a single reverse primer (denoted as Rn with n being the number of the set) and a varying number of forward primers (denoted as Fn.m followed by the induced substitution). Every set covers a small range of amino acids in the DNA-binding domain of BlcR, with each position being mutated to different residues by different forward-facing primers. Do note, however, that while a large number of primers have been designed, sequencing showed that the mutagenesis protocol was not successful for all variants. Therefore, only the primers that resulted in the desired mutants will be described in our collection of BioBricks.
The primer sets are divided as follows:
- Set 1, residues 36 to 39
- Part:BBa_K4361200: R1
- Part:BBa_K4361201: F1.1 D37R
- Part:BBa_K4361202: F1.2 D37V
- Part:BBa_K4361203: F1.3 L38V
- Set 4, residues 40 to 41
- Part:BBa_K4361204: R4
- Part:BBa_K4361205: F4.1 A40V
- Set 5, residues 47 to 49
- Part:BBa_K4361206: R5
- Part:BBa_K4361207: F5.1 T47S
- Part:BBa_K4361208: F5.2 T47V
- Part:BBa_K4361209: F5.3 A48V
- Set 7, residues 61 to 63
- Part:BBa_K4361210: R7
- Part:BBa_K4361211: F7.1 S61V
- Part:BBa_K4361212: F7.2 A62V
- Part:BBa_K4361213: F7.3 A62I
- Part:BBa_K4361214: F7.4 A62K
- Part:BBa_K4361215: F7.5 A62T
- Part:BBa_K4361216: F7.6 H63V
- Part:BBa_K4361217: F7.7 H63Y
- Set 3, residues 65 to 68
- Part:BBa_K4361218: R3
- Part:BBa_K4361219: F3.1 L66V
- Part:BBa_K4361220: F3.2 L66A
- Part:BBa_K4361221: F3.3 L66I
- Part:BBa_K4361222: F3.4 A67Q
- Part:BBa_K4361223: F3.5 A67V
- Part:BBa_K4361224: F3.6 A67H
- Part:BBa_K4361225: F3.7 V68T
- Part:BBa_K4361226: F3.8 V68K
- Part:BBa_K4361227: F3.9 V68S
This primer contains a mutation that changes the codon for alanine (A, GCG) in position 62 to that of lysine (K, AAA).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]