IGEM:Hong Kong HKUST/Investigations/ Plasmid Cloning by Restriction Enzyme Digestion /Entry Base
This is an investigational project aimed at providing one with essential skills to carry out cloning involving 2 restriction enzymes. The entire training duration starts from inoculations of bacteria and ends with ligation products' transformations.
The plan for this project is to swap the pTET fragment from (BBa_R0040) which is 54bp in length, with the entire red fluorescent protein (RFP) reporter coding sequence from the construct pSB1C3_BBa_J04450 which is 1069bp in length back into the BBa-R0040 vector backbone (pSB1C3). Firstly the RFP coding sequence from (BBa_J04450) needs to be cut from (pSB1C3_BBa_J04450). Then the RFP CDS is cloned back into the multiple cloning site of linearised pSB1C3. The ligation product is then transformed into E. coli competent cells. The positive clones should be phenotypically red in colour.
Materials and Methods
Single colonies of bacteria harbouring biobricks BBa-R0040 and pSB1C3_J04450 were inoculated with 5mL of Luria Broth supplemented with 34μg/mL chloramphenicol (LB) medium in 6 x 50mL autoclaved falcon tubes (3/bacterium) and grown overnight in a 37°C incubator.
DNA Extraction and Digestion
The extraction of the plasmid was done according to the MiniPlus DNA Extraction Kit protocol available here. Post extraction, the concentration of the plasmid was checked with the nanodrop 2000 machine from Thermo Scientific NanoDrop machine.
2 μg of each plasmid were digested with 0.5 μL of EcoR1-HF and 0.5 μL SpeI-HF in buffer 4, and diluted accordingly until the total reaction volume was 50 μL. Overnight digestion was carried out at a 37°C incubator
PCR was done according to the New England Biolabs protocol here in 25μL reactions with standard biobrick primers VF2 and VR. The master mix for 14 reactions were made and divided into 12 tubes with each containing 25 μL. Then the amplicons were analysed with gel electrophoresis.
Gel Purification and Ligation
Following the overnight digestion, on the next day, gel purification was done with a 1% gel. After confirming the lengths of the digest DNA fragments, the gel with the DNA of interest was cut and purified according to FavorGen Gel/PCR Purification Kit. Once again, the concentration was measured with a nanodrop 2000 machine NanoDrop machine. Later on all the parts ligated together with T4 Ligase enzyme overnight at 16 °C as suggested here
The transformation was done following this protocol here. The transformed bacteria were spread evenly to the agar plates supplemented with 34μg/mL chloramphenicol.
The total colonies on Bosco's plate are 437 with 430 of them are red. Therefore 98.4% of Bosco's clones are positive clones. While the total colonies on Felicia's Plates are 220 with 218 of them are red. Therefore 99.1% of Felicia's clones are positive clones.
The phenotype of our positive clones is red, so the positive colonies are easily distinguished. This is the reason why we choose BBa_J04450 for our project, so that we can easily count the number of the positive clones. We have reached a high efficiency because of the overnight digestion and ligation in order to make sure the processes are completed before the following procedure. We also used unique digestive enzymes, which are EcoR1 and SpeI, to make sure there is no self-ligation of the vector backbone. The ratio of the backbone and the insert also adjusted to 1:3 to make the efficiency higher as well.
As for the PCR practice with both BBa-J04450 and BBa-R0040 as the templates with primers VF2 and VR, there were bands of the desired size. The PCR failed with no apparent reason despite all the components were added appropriately. The next day post PCR, Raul found that the PCR machine was broken and could not be switched on.
We have successfully swapped the pTET promoter in BBA-R0040 with an RFP Coding Device.