Team:Peking/Notebook
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Experimental principles
Between the mutual transformation of music and DNA, the most important thing is to introduce mutations. In addition to computer simulation, we also carried out a series of laboratory work to further demonstrate our mutation results.
First, we thought of making CRISPR/Cas9 based mutagenesis enzymes directly work on raw information sequences. However, we may come to a stop a few generations later when sgRNA could not recognize its target anymore. We either end up with rarely mutated sequences or have to adjust sgRNA itself, neither of which would be a good result. Thus, we come up with the idea to separate each sequence into two parts. One is the information sequence which stores actual music information, and ahead of it is the anchor sequence where sgRNA binds.
The sequence of each section consists of a coding region and an anchor interval. The anchor interval is targeted by the gRNA, and then the coding sequence is mutated by the base editor. Because the mutation accuracy of the base editor is not 100%, it may mutate the adjacent sites of the mutation site. We chose early base editors (less accurate and more prone to random mutations), and simultaneously introduced them into E. coli along with our music coding sequence and separate gRNA expression plasmids. The base editors included both ABE (adenine base editor) and CBE (cytosine base editor), so that their combination can change both A-T and C-G base pairs. In this way, we can carry out large-scale random mutations in the coding range of the whole music coding sequence, and then re-sequence these mutated sequences. By using our algorithm, we can re-import the high scoring sequences into E. coli, and then repeat the cycle, and we can get the mutation sequences with higher scores.
Introduction of EvolvR
Introduction of ABE and CBE
Lab notebook
| Date | Process |
|---|---|
| 7.29 | Cloning of plasmid I0050, J23119, B0015 with pBAD, RBS, ddTerm part respectively / Extracting a small number of plasmids and measuring the concentration |
| 7.30 | Selection of bacteria with various components / Enzyme digestion and ligation of pBAD, ddTerm, RBS and PSB1C3 by BioBrick |
| 8.3-8.4 | Preparation and split charging of chloramphenicol / Enzyme digestion and ligation of 1C3-ddTerm, B0015, 3C5-pBAD-dcas9-J23119-sgRNA, pBAD-dcas9-J23119-sgRNA / Transforming and amplifying / Extracting a small number of plasmids |
| 8.5-8.8 | Build ABE, CBE part with 15 AA linker and 10 AA linker / Enzyme digestion and ligation of plasmids / Extracting a small number of L-pmCDA-I-UGL-I (Amp+) |
| 8.9-8.10 | Ligation of sgRNA scaffold by ePCR / PCR was used to verify ABE-dCAS9, CBE-dCAS9 and sgRNA scaffolds |
| 8.12-8.13 | Each element was cut off from PUC57, incluing UGI, CDA+UGI, pBAD / Construction of pBAD+RBS, pBAD-long+RBS / PCR was used to verify pBAD+RBS, pBAD-long+RBS, TadA, dCAS9 |
| 8.14 | Amplifying PSB1C3-pBAD-RBS, Puc57-ABE-UGI, Puc57-CBE-UGI, PSB1C3-sgRNA scaffold / The bacterial liquid was verified by PCR and sequencing |
| 8.15 | The EvolvR system was divided into five fragments with more than 1000bp, which could be connected with each other through Bsa1 and synthesized by the company |
| 9.29 | All the plasmids mentioned before were of low concentration and needed to be re extracted / Retransform PSB1C3-pBAD-RBS, Puc57-ABE-UGI, Puc57-CBE-UGI, PSB1C3-sgRNA scaffold |
| 9.30 | Most of the colonies did not grow and the original bacterial solution should be resuscitated and coated |
| 10.1 | Extracting a small number of plasmids pSB1C3-pBAD-RBS puc57-ABE-UGI puc57-CBE-UGI pSB1C3-sgRNA scaffold and measuring the concentration / Verified by PCR, Most of them have problems |
| 10.2 | Reusing another batch of preserved plasmids and transforming plasmidsp SB1C3-pBAD-RBS puc57-ABE-UGI puc57-CBE-UGI pSB1C3-sgRNA scaffold |
| 10.3-10.4 | There are some problems in PSB1C3-sgRNA scaffold and PSB1C3-sgRNA scaffold / 5 parts of EvolvR was coated and extracted / Retransforming PSB1C3-sgRNA scaffold, PSB1C3-sgRNA scaffold |
| 10.5-10.6 | PSB1C3-sgRNA scaffold and PSB1C3-sgRNA scaffold were verified successfully by PCR / Puc57-ABE-UGI puc57-CBE-UGI carriers were linearized by BioBrick but lost with plasmids in accident |
| 10.7-10.8 | Restart (see 10.2) |
| 10.11 | Verifying by PCR / There are some problems in PSB1C3-pBAD-RBS and restart transforming it. |
| 10.12-10.13 | PSB1C3-pBAD-RBS was verified successfully by PCR / Puc57-ABE-UGI, Puc57-CBE-UGI, PSB1C3-sgRNA scaffold were digested by enzymes and linearized / PBAD-RBS was attained / Amplifying PSB1C3-ddTerm and EvolvR parts |
| 10.14 | We got ABE-final and CBE-final by ligating Puc57-ABE-UGI, Puc57-CBE-UGI and ddterm, PBAD-RBS |
| 10.15 | EvolvR parts were digested from the synthesized vector and PSB1C3 was digested at BSA1 site / Ligating them |
| 10.16-10.17 | ABE-final, CBE-final, EvolvR-final / PCR confirmed that it was positive |