DNA

Part:BBa_K1732011

Designed by: Donna Lee   Group: iGEM15_Carnegie_Mellon   (2015-09-09)

pSB1C3-J23100-B0034-OFP-His-B0015

His tagged OFP and strong RBS. The J23100 strong constitutive promoter allows for high level expression of the fluorescent protein used to measure fluorescence.


A set of fluorescent proteins which includes BFP, GFP, eGFP, E0040, OFP, YFP, and RFP with a J23100 constitutive promoter and RFP with a J23115 constitutive promoter were characterized. The relative fluorescence as well as the signal-to-noise ratio of all the proteins in the MACH competent cell line were calculated. OFP was measured at (ex/em = 548nm/562nm).


FIntensity.jpg

FQuantitation1.jpg



Excitation and emission scans were performed on triplicate readings using the Tecan M1000. Fluorescence readings are normalized and shown below. Maximum fluorescence was found at ex/em = 546nm/562nm.

Cell growth of overnight cultures diluted by a factor of 50 was measured every 30 minutes for 6 hours. OD660 readings are shown below.

Fluorescence of the diluted cultures was read every 30 minutes for 6 hours at (ex/em = 548nm/562nm).


Alignment

Sequence alignment of original sequence to codon optimized sequence, indicating the differences in amino acids.

Alignment OFP.jpg



Original Set of Fluorescent Proteins (Not Codon Optimized)

The graphs below show the data for the original set of fluorescent proteins prior to being codon optimized via the IDT codon optimization tool. The set consists of His tagged RFP, OFP, YFP, eGFP, and BFP, each with a strong RBS and WT lac promoter that allows for Lac induction of fluorescence.

Fluorescent proteins characterization.jpg

Here we take various dilutions of each extracted protein and using their concentration from above we provided an exact amount for each. We graph amount versus total fluorescence as read with a gain of 100 on a TECAN Saffire2 fluorimeter. as shown by the r value, it appears that there is a linear relationship between fluorescence and quantity of protein with two exceptions: RFP and YFP. RFP appears to increase logarithmically, whereas YFP has a negligible protein output due to low expression and possibly repeated error in extraction. It is important to note that despite low quantity, YFP remains highly fluorescent.

Fluorescent proteins Amounts.jpg

This graph quantifies fluorescent proteins using a BCA protein asset with a BSA standard. The proteins were His-Tagged and extracted using a Ni-NTA protocol. from there we used a spectrophotometer to create a standard curve with BSA of known concentrations and a BCA dye. The dye allowed us to read the amount of protein from 1.5mL of culture as a function of absorbance (in a sense amplifying the scale of our readout for a reading more accurate than obtainable in the UV range with our available equipment).


Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 631
  • 1000
    COMPATIBLE WITH RFC[1000]


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