Week 10 HW: ADVANCED IMAGING & MEASUREMENT TECHNOLOGY

1. Final Project

I will measure whether DNA stored on paper can be recovered and remain readable after storage. In Aim 0.5, the main measured output is the presence or absence of expected PCR bands from recovered DNA fragments. Fragment A is expected to produce a ~94 bp payload PCR product, and Fragment B is expected to produce a ~166 bp product. I measured this using endpoint PCR followed by 2% E-Gel EX agarose gel electrophoresis.

I will also compare DNA recovered from APTMS-treated paper and untreated paper. In the current pilot, this comparison is qualitative: whether each condition produces the expected band. In future work, I would measure recovery more quantitatively using qPCR Ct values, digital PCR copy number, or fluorometric DNA quantification.

For the next stage of the project, I would measure whether recovered SspCA-encoding DNA can drive functional protein production in a cell-free expression system. This would include measuring SspCA protein expression, expected protein size, and carbonic anhydrase activity. Possible technologies include SDS-PAGE or Western blot for protein size and expression, fluorescence or colorimetric assays for enzyme activity, and DNA sequencing to confirm that recovered DNA remains sequence-correct.

Technologies used or proposed:

  • Endpoint PCR: tests whether recovered DNA remains amplifiable.
  • E-Gel EX agarose gel electrophoresis: visualizes expected DNA bands and confirms approximate amplicon size.
  • qPCR or digital PCR: future quantitative measurement of DNA recovery.
  • DNA sequencing: future verification of sequence integrity and encoded data.
  • Cell-free protein expression: tests whether recovered DNA can produce SspCA.
  • SDS-PAGE / Western blot: verifies protein expression and approximate size.
  • Carbonic anhydrase activity assay: measures whether produced SspCA remains functional.

2. Waters Part I

Q1:The sequence provided includes the eGFP core, an LE linker and a 6x-His purification tag. Calculated Molecular Weight: 27,988.97 Da

Q2:

  • Peak 1 (m/z_n): 875.4421

  • Peak 2 (m/z_n+1): 903.7148

  • z for each adjacent pair of peaks: the charge state for the 903.7 peak is 31+, and the 875.4 peak is 32+.

  • MW of the protein: 27,981.90 Da

  • Accuracy of the measurement: 252.6 ppm

Q3: No.

3. Waters Part II

Q1:

Denatured state: The protein unfolds because of heat or chemicals. More amino acids become exposed, so the protein gains more charges and shows lower m/z values.

Native state: The protein stays folded in its normal shape. Fewer amino acids are exposed, so the protein gains fewer charges and shows higher m/z values.

Q2:

Yes. The charge state is 10+. The isotope peaks are spaced by 0.1 m/z, and since spacing = 1/z, 0.1 means z = 10.