Difference between revisions of "Part:BBa K3229302"

Revision as of 17:08, 21 October 2019

McyA promoter reporter (with reverse promoter)

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 reverse McyD promoter(McyA sense), RBS, GFP and a terminator sequence.

(konstrukt kép)

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 bacterias 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 bacteria that has the insert containing ligated plasmid will outgrow.

(Kép a vectorról)

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.

(Kép az electroforézisről)

On the above picture we can see the result of the running. We can see the vector at the fourth line, because its length is 3519 bp. Between the sixth and seventh line we can wee out inserts, their length is 1741 bp and 1829 bp (A/B, C/D respectively)

Sequence and Features