Synthetic Biology

If the mRNA molecule shown is produced inside the blue cells using a biological circuit what will happen ?

The mRNA-encoded protein won't be translated

Which part of a biological circuit is necessary for a gene to be transcribed?

Promoter

Ok, so of the promoter comes first and that initializes transcription, what do you think the next downstream component on the plasmid is ?

Coding sequence

With this in mind, we should add a sequence that is...

... complementary to miRNA that has low expression in cancer cells but high expressions in healthy cells

Which of the following sites is cut by BP clonase mix enzymes ?

attB1

What outcome do you predict if you mix the following plasmid and linear DNA fragment with BP clonase enzymes ?

Outcome b

What limits the number of parts that can be cloned into a single expression vector with the Gateway reaction

The nnumber of different att sites

What feature links the different circuit parts together

att sites

Which Gateway reaction produced the att sites in these plasmids ? Think about which type of site is converted to the ones shown above

BP

To take advantage of the Gateway reaction, what additional sequences should we add to each DNA fragment before ordering?

att sites

Which att sites does the additional entry vector need to contain to assemble all the circuit parts into the destination vector?

attL1 and attR2

Why does the BP clonase mix only produce attL and attR sites ?

It specifically cuts attP and attB sites

What att sites should be therefore add onto the linear sequences we are ordering ?

attB1 and attB2

Why is it important to keep the clonase enzymes on the cooling rack at this point ?

The enzymes degrade more slowly when kept chilled

What is pENTR_L4_BAX_R1? Remember the naming conventions for different vectors you learned about earlier today ?

An entry vector with the BAX gene sequence, flanked by attL4 and attR1 sites

What is NOT a feature of the destination vector?

BAX killer gene

How can we find the cells that were transformed with our vector ?

Grow the cells on a growth medium containing antibiotics

Our tube contains hundreds of cells that took up different vectors. Which is certainly NOT contained by any live cell?

pDEST

Which antibiotic should we use to select for cells containing the expression vector?

Ampicillin (Amp)

How are you going to spread them ?

With a sterile tool

Which plasmid do the surviving cells on the agar contain?

none of these options

Which mix contains enzymes that can cut DNA and produce stick ends?

Gateway

Which of the following restriction enzymes would you use to perform digestion?

Xbal

How many DNA fragments do you predict will be produced by digesting it with Xbal?

1

What does the pattern indicate?

The construct is part of the plasmid