Part:BBa_K1429002

Cyan Fluorescent Protein (CFP) "Cindy Lou" coding region, intellectual property-free

IP free cyan fluorescent protein. We have a ribosome binding site (RBS).

Background

We want to take Tuesday's PCR products and put them into the pSB1C3 backbone. 

Digest PCRs:

10 ul PCR product

2 ul cutsmart buffer (10x stock)

1 ul PstI

1 ul EcoRI

20 ul total --> incubate for 30 min at 37C

 

PCR purify digest product (only 14 ul - save 6 ul):

Follow kit protocol. Elute in elution buffer.

Worried that the washed columns won't bind DNA, we are going to use some of the set-aside (unpurified) digest product for a backup ligation. We'll run a gel of our purification, but we are going to set up a ligation beforehand, so we won't have even rough estimates of DNA concentrations.

Set up ligations:

<tbody> </tbody>

Component	Using purified digest product	Using unpurified digest product	BB alone
dH2O	x	11	14
Insert (RFP or GFP)	14	3	x
1:10 BB	3	3	3
T4 buffer (10	1	1	1

The above were incubated 30 min at RT then stored at -20C.

 

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Results

We ran a 1% gel of the digest before and after purification. We had a decent yield, maybe 40% of our initial digest product in the purified lanes. 

Conclusions

Next step: Transform the ligated plasmids into E. coli!

Background info:

• The plasmid is about 2000 base pairs (bp).  
• Our PCRed GFP (gene of interest) is about 750 bp.
• Assume that 1 uL of this PCR purified GFP = 50 ng (info from Ellen).

Calculations:

We have 6 uL of our gene of interest in a solution totaling 20 uL.

So we have:

\(\displaystyle \frac{6\mu L }{1}PCR \; purified \; GFP * \frac{50 ng}{1 \mu L} * \frac{1}{20\mu L} = \frac{300 ng}{20 \mu L} PCR \; purified \: GFP = 15\frac{ng}{\mu L}PCR \; purified \; GFP\)

 

The iGEM kit provides a linearized plasmid backbone in a solution with a concentration of 25 ng/uL.  

In our digestion step, we used 4 uL of the linearized plasmid backbone and 4 uL of the enzyme master mix. 

So, we have:

\(\displaystyle \frac{4\mu L }{1}plasmid * \frac{25 ng}{1 \mu L} * \frac{1}{(4+4)\mu L} = \frac{100 ng}{8 \mu L} plasmid= 12.5\frac{ng}{\mu L}plasmid\)

We need to have a GFP:plasmid ratio of at least 3:1 to make sure that we have enough pieces of the gene of interest to successfully connect to the plasmid backbone.

Now, we'll use the <a href="http://nebiocalculator.neb.com/#!/" target="_blank">NEBioCalculator</a>  to get the number of moles for each (see the pictures).

So, we have:

\(\displaystyle \frac{32 . 36 \frac{fmol}{\mu L} \; GFP}{9 . 632 \frac{fmol}{\mu L} \; plasmid} = 3 . 36 \; GFP : 1 \; plasmid \gt 3 \; GFP : 1 \; plasmid\)

Therefore, we can use a ratio of  1 uL of the PCR purified GFP solution to 1 uL of the digested plasmid solution.

 

Sequence and Features

Assembly Compatibility:

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

  Illegal XhoI site found at 74
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 2