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Design and ordering of DNA parts
- Ordering of parts from GENEART: We have ordered 11 gene sequences from GENEART. We have synthesized pCooM, pCooF promoter and its 4 variants, CooA protein and 4 of its variants. Those genes are proven to be successful in cloning.
- Design of parts: We have designed two different vectors , one containing pTriEx vector containing CooA and one containing our final construct. However, we couldn’t observe fluorescence with this system because of high expression capability of pTriEx. Because of this factor, we have designed another biobrick containing pLacI-RBS-RFP. We have cloned this with pCooM-RBS-RFP-TT and pSB1C3. We didn’t have enough time for cloning of the final vector with this biobrick.
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'''Cloning'''
- Preparation of competent cell: 2 different methods and 1 kit were used for competent cell preparation. Fermentas Transformation kit wasn’t successful at all. Although two different TOP10 Glycerol stocks were used for this procedure, the preparation of competent cells couldn’t be accomplished. After working with Fermentas kit, TSS and Rubidium Chloride methods have been tried for competent cell preparation. Rubidium chloride method was chosen for competent cell preparation
- Visualizing Small DNA fragments on agarose gel: For this purpose, two different methods have been utilized. Firstly, ethanol precipitation method was used to concentrate DNA samples prior to assembly of biobricks. Although this method was successful, owing to the fact that it has time constraints, Vacuum drying method was used for this purpose. By this procedure, we have successfully concentrated our DNA samples.
- Restriction Digestion: Two different enzyme types of Fermentas have been used for this purpose (Fast Digest-Conventional).Fast Digest enzymes have proven to be more useful because of time saving properties.
- Ligation: Ligation procedure was performed according to molar ratio of fragments.
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Validation'''
- Sequencing: 5 of parts have been sequenced and 1 one the parts was completely wrong sequence. PcooM-RBS-RFP–TT part doesn’t have the right sequence. For this reason, we have cloned this part again and sent to IGEM headquarters. However, we didn’t have enough time for sequencing of other parts.
- Agarose gel Electrophoresis: We have observed exact bands on agarose gel for each part.
- Fluorescence Spectrophotometer and Confocal Laser Scanning Microscopy data: Experiments done with pCooM-RBS-RFP-TT-pLac-RBS-CooA showed that RFP fluorescence is very high with CO applied samples. Those results show that our newly cloned biobrick works successfully.
- Agar plates: Our Agar plates with pCooM-RBS-RFP-TT-pLac-RBS-CooA showed that because of low level of constitutive binding of CooA to pCooM promoter, we have observed low level of RFP fluorescence on agar plates.
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Assembly of final construct'''
- Construction: We have designed two different vectors, one containing pTriEx vector containing CooA and one containing our final construct. Our final construct shows to be ineffective when there is pTriEx expression vector. Owing to this, we have designed a new vector which has pLacI-RBS-CooA. However, we didn’t have enough time for complete construction of the final vector.
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