Cloning proyect 1. What does constitutively active promoter mean?

Why would you want to use a vector with this type of promoter? It is a promoter that is not regulated by any elements allowing continues transcription of its gene. Having a constitutive promoter in a vector will permit the transcription of rscB gene without concerns for gene regulation, which boost RscB expression. 2. Using the protein sequence of RscB in Y. enterocolitica, do a BLAST search to determine if homologs exist in other organisms. If so, what are your top hits (just name 2 or 3)? Rsc B its though to be a surface expose protein. A BLAST search for RscB of Y. enterocolitica showed homology with a hemolysin activation/secretion protein associated with VreARI signaling system and with a supposed lipoproteins in Y. pestis. STRING, a web protein-protein prediction system, predicts interacions with hemolysin and transporter proteins. Hemolysins are proteins that provoke red blood cells lysis. For bacteria, this proteins are important for nutrient acquisition, special iron.
3. Design and outline a strategy to determine the direction that your gene has been cloned into the TOPO vector. Your strategy should include how you would test this as well as an outline of your expected results if your gene is in one direction or the other. Be sure to look up the enzymes that you will be using to do this and outline your protocol with details necessary for you to set up this reaction in a lab.

i) In order to determine the rscB gene direction into the TOPO vector a digestion reaction will be develop. Using the enzymes BbsI and BamHI-HF, digest the DNA. The product of this reaction will be two DNA sequence. If the vector is in the forward direction, one band of ~4.6kbp and one band of ~309bp would be seen when a 2% agarose gel is run (Figure 1). In the other hand, if the rscB gene is the reverse direction, one band of ~4.2kbp and one band of ~719bp would be reflected in the agarose gel after the electrophoresis (Figure 2). In addition, positive and negative controls are highly recommended. ii) Rx set up: (1) Digestion Rx: 36 uL water, 5.0uL BSA, 5.0 uL NE Buffer 1, 1.5 uL DNA (500ng of DNA to observe at ~30-80ng of DNA in the smaller possible band), 2.0 uL Bbs 1 (10 units), 0.5 uL BamHI-HF (10 units). (2) Negative control: 38.5 uL water, 5.0uL BSA, 5.0 uL NE Buffer 1, 1.5 uL DNA. (3) Positive control for Bbs 1: 36.5 uL water, 5.0uL BSA, 5.0 uL NE Buffer 1, 1.5 uL DNA, 2.0 uL Bbs 1. (4) Positive control for BamHI-HF: 38 uL water, 5.0uL BSA, 5.0 uL NE Buffer 1, 1.5 uL DNA, 0.5 uL BamHI-HF. iii) Run the reaction for at least 1hr @ 37 C.

iv) Run a 2% agarose gel electrophoresis. Load 5uL with 1X loading dye (1uL from 6X) from each reaction into a well. v) Expected results Figure 1

Figure 2

4. Design and outline a strategy to move your gene into the TOPO vector. Your strategy should include the restriction enzymes that you would use to move your rscB gene into the pWKS130 vector (taking into account the direction it is in the TOPO vector), as well as a strategy to verify you have cloned it in the proper direction in the pWKS 130 vector and your expected results. Be sure to look up the enzymes that you will be using to do this and outline your protocol with details necessary for you to set up this reaction in a lab.

i) Depending on the results from agarose gel, you will pick to digest for the forward or reverse direction in order to have the correct sequence of the start codon next to the promoter in the pWKS130 vector. (1) TOPO + insert digestion: (a) Digestion for forward: 1hr @ 37 C 1. BamHI-HF: 0.5 uL 2. Xbal: 0.5 uL 3. DNA (TOPO + insert): 10 uL (10000ng ~1800ng of Insert) 4. 1X Ne Buffer 4: 5 uL 5. 1X BSA: 5 uL 6. dd water: 29 uL (b) or Digestion for reverse: 1. Xho 1: 0.5 uL 2. BamHI-HF: 0.5 uL 3. DNA (TOPO + insert): 10 uL (10000ng ~1800ng of Insert) 4. 1X Ne Buffer 4: 5 uL 5. 1X BSA: 5 uL 6. dd water: 29 uL (2) pWKS130 vector digestion: 1hr @ 37 C (a) Digestion for forward: 1. BamHI-HF: 0.5 uL 2. Xbal: 0.5 uL 3. DNA: 4uL for 1000ng 4. 1X Ne Buffer 4: 5 uL 5. 1X BSA: 5 uL 6. dd water: 35 uL (b) or Digestion for reverse: 1. Xho 1: 0.5 uL 2. BamHI-HF: 0.5 uL 3. DNA: 4 uL for 1000ng 4. 1X Ne Buffer 4: 5 uL 5. 1X BSA: 5 uL 6. dd water: 35 uL ii) Run 5 uL of the digestion product in a 5% agarose gel electrophoresis. This will run ~180ng of the insert DNA in the gel. To dilute the DNA add 20 uL of dd water. iii) Isolate the correct bands. ~900bp for insert iv) Purify the DNA band on the gel following the QUIK gen purification protocol. v) Quantify the DNA concentration.

vi) Ligation: ligate the insert into the pWKS130 vector in a 3:1 ratio. (1) Add 16 ng of vector to 50 ng of insert, and adjust the volume for 10 uL with ddwater. (2) Add 10 l of 2X Quick Ligation Buffer and mix (3) Add 1l of Quick T4 DNA Ligase and mix (4) Centrifuge briefly and incubate at room temperature (25C) for 5 minutes. (5) The ligation product is ready to use. Store for until use on ice.