|
|
| Line 1: |
Line 1: |
| | | | |
| - | <p>The work of mutagenesis group focuses on the construction and tests of mutagenic device. The mutagenesis plasmid’s structure has </p><p>been described in Design-Mutagenesis and we specifically aim to confirm that:</p>
| |
| - | <p>1）All functional genes mutagenic genes can be expressed in our system.</p>
| |
| - | <p>2）This device can remarkably increase the mutation rate of target sequence. </p>
| |
| - | <p>3）(In the future) The functionality of this device can be activated or deactivated at proper stages, and work in tune with B2H </p><p>system and M13 proliferation cycle, further conducting directed revolution.</p>
| |
| - |
| |
| - | <p>Our work are as follows:</p>
| |
| - |
| |
| - | <p>Obtaining functional genes</p>
| |
| - | <p>The four primitive genes dnaQ, umuD, umuC, and recA are obtained from genome of E. coli BL21 (DE3) by PCR and transferred into pMD-</p><p>19T. The reason for using pMD-19T is to make the following procedures easier to be carried out. Using quick-change PCR (see Fig-2), </p><p>we conducted point-mutations on dnaQ and recA and obtained dnaQ926 (D12A, E14A) and recA730 (E39K). For recA730, two synonymous <p>mutations were performed additionally to delete the inherent restricted enzyme recognition sites EcorI and PstI. UmuD’ was the N-<p>term (4-72bp) truncated umuD.
| |
| - | </p><p>We checked the plasmids by PCR to make sure functional genes have been inserted. The agarose gel electrophoresis result is showed <p>below. All genes have been confirmed by sequencing.
| |
| - | </p><p>Expression test of mutagenic genes
| |
| - | </p><p>After we had got the four mutagenic genes, plac (BBa_R0010) was chosen as the promoter in our expression vectors. RFP was used to </p><p>test whether genes were expressed. Specific operation method is to add RFP (BBa_E1010) behind single mutagenic gene and transfer the </p><p>plasmid into BL21. Testing RFP fluorescence (480nm/510nm), the positive index indicated the successful expression of our genes.
| |
| - | </p><p>The plasmid structures are as follows:
| |
| - | </p><p>plac + RBS (BBa_B0034) + mutagenic gene + RBS + RFP (BBa_E1010)
| |
| - | </p><p>We planned to express four mutagenic genes on one single plasmid, so we also constructed a test vector as follows:
| |
| - | </p><p>plac + RBS + mutagenic gene + RBS + mutagenic gene+ RBS + mutagenic gene+ RBS + RFP
| |
| - | </p><p>Pure liquid LB broth was chosen as blank control.
| |
| - | </p><p>In the future, we plan to test these mutagenic genes combined with more inducible promoter pbad (BBa_I13453) and RBS with different </p><p>intensities.
| |
| - |
| |
| - | </p><p>Test of evolutional mutation
| |
| - | </p><p>All together, the four mutagenic genes should induce obvious DNA mutation. In order to test such mutation, we use RFP as mutation</p><p>target. As showed in Fig-3, we constructed the mutagenesis plasmid, pSB1A2 with similar Plac, RBS and all four genes as polycistron.</p> <p>The reporter plasmid to be mutated is pSB1C3 with constructive promoter BBa_J23100, RBS and RFP. Then we co-transformed both</p> <p>plasmids into E. coli ER2738, a strain able to be infected by M13. After cultivation of positive red strains in LB (with Amp and Cm)</p> <p>solution, the diluted bacteria was spread on LB plate with Amp and Cm. If white strains, rather than red, are observed (which </p><p>indicates RFP chromophore is mutated), pick out the white strains and sequence the promoter and RFP to confirm whether it is exactly </p><p>mutated.
| |
| - |
| |
| - | </p><p>Module and simulation of the mutation rate
| |
| - | </p><p>For the DNA polymerase III proof-readingε-subunit, DNAQ926 works by competition mechanism. We have constructed a module to simulate</p> <p>the mutation rate when the two kinds of subunits (wild-type and mutant-type) both exist in E.coli.
| |
| - | </p><p>As reported, the mutation rate of well-functioned DNA polymerase is〖10〗^(-10), while that of the proofreading-defected one is </p><p>〖10〗^(-5).For there is no significant functional difference between the defective and non-defective polymerase but mutation rates,</p> <p>we calculate the average mutation rate with formula as follows:
| |
| - | </p><p>We assume that the number of DNAPIII is 15 as it’s ranged from 10 to 20 in E.coli. The mutation rate changes as below when the </p><p>number of defective polymerase varies from 1 to 40.
| |
"
