Part:BBa_K3229301
McyD promoter reporter
This part is appropriate to measure the activity level of the putative mcyD promoter region. Normally we can find toxin-forming protein genes (McyABC and McyDEFGHIJ) next to the promoter in both 5'-3' and 3'-5' directions, thus the promoter can regulate the formation of the microcystin toxin through the synthesis of microcystin producing proteins. When the toxin is present it enhances the transcription from the promoter due to the phenomenon called autoinduction. We replaced the toxin-producing protein genes with GFP, thus when microcystin appears it induces the production of GFP. We analyze the emitted light with a fluorescence detector, which is widely used among high schools, and decide whether the fluorescence is correlated with the toxin concentration. This part consists of the McyD promoter (McyD sense), RBS, GFP and a terminator sequence.
For cloning we used the Zero Blunt TOPO PCR Cloning Kit from Thermo Fischer Scientific (https://www.thermofisher.com/order/catalog/product/450245?tsid=Email_POE_OC_OrderConfirm%20%0D%20_SKULINK#/450245?tsid=Email_POE_OC_OrderConfirm%20%0D%20_SKULINK). It uses a vector, called pCR-Blunt II-TOPO. The ligation of the vector and insert is done by topoisomerase enzymes, which are connected to the ends of the linearised vector. At the 5’ side of the cleavage there is a Plac promoter, which starts transcription from lacZalpha gene (which is located at the 3’ side of the cleavage). To the C-terminus of the lacZalpha gene a lethal ccdB gene is connected. If the ligation is succesful it interrupts the transcription from the lethal gene, thus promotes E. coli growth. To sort the bacteria which do not have the plasmid, there is a Kanamycin resistance gene in the vector. If we spread the bacteria on Kanamycin containing LB only the ones that have the insert containing ligated plasmid will outgrow.
We used gel electrophoresis to check if the bacteria had the right plasmid inside them. Before running, we digested the purified plasmids with NotI restriction enzyme. This digestion leaves us with inserts and vectors separately, because the insert had NotI containing prefix and suffix at the ends. We run them on 110 V for 30 minutes. We used lambda DNA digested with EcorHI and HindIII marker as a ladder.
On the above picture, we can see the result of the running. The vector is near the fourth line from above and the inserts are between the sixth and the seventh line. The original lengths were 3519 for the vector, 1741 for A/B and 1829 for C/D. This proves that the plasmid, which was transformed into the bacterial cells, contained our inserts.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 578
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1565
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