Part:BBa_K3229304

McyA/D biderectional promoter 2

This part is constructed to certify the bidirectional transcription from McyD promoter. Normally microcystin toxin-forming protein genes (McyABC and McyDEFGHIJ) are located next to the McyD promoter in both 5’-3 and 3’-5’ directions. When microcystin toxin is present, it enhances the synthesis of these proteins, thus promotes more toxin production. This phenomenon is called autoinduction.

We replaced the toxin-producing genes with a composite GFP, which consists of two parts. We put one part in the 3' direction and the other one in the 5' direction from the McyD promoter. When the two parts are synthesized at the same time they connect together to form an active GFP that glows. Thus The transformed bacteria that have the insert can only glow under UV light if the bidirectional transcription works. This construct consists of the McyD promoter, an RBS, GFP2 and a terminator sequence in the 3' direction from the promoter and a reverse RBS, a reverse GFP1 and a reverse terminator sequence in the 5' direction from the promoter.

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1108
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1622