Team:BostonU/FusionProteins

June

Week of June 23

• Used Phusion PCR to make the parts listed above and then, performed PCR cleanup to purify the DNA fragments.
• Upon quantifying all clean-ups, I found that the concentration for the E0040_ID (4.4 ng/uL) cleanup was lower than that of the negative control (6.1 ng/uL). So, I repeated the Phusion PCR for that part and ended up with a concentration of 31.9 ng/uL.
• Performed MoClo Level 0 reaction to insert the purified constructs into backbones with a Cam resistant gene.
• Transformed the MoClo plasmids on Cam plates to perform Blue-White screening and pick colonies that had successful digestion-ligation
• Transformation didn't work because I used faulty Bioline cells. So, I repeated transformations using DH5a cells.
• Screened colonies further by performing Colony PCRs and running a gel.

Week of June 30

• Ran gel for previous PCR reactions
  1. Two terminators were too small to see well on gel
• PCR in triplicate for all genes and terminators as well as negative control
• PCR clean-up
  1. Didn't work for ECK120015170 because it was too small & fell through the filter
• Level 0 MoClo reactions for BM3RI, BetI, SrpR, LmrA, L3S2P21
• Redid PCR for ECK120015170
• Level 0 MoClo reaction for ECK120015170
• Transformations for all Level 0 MoClo reactions
  1. None of the transformations worked
  2. Accidentally used Pro cell strain with Cam resistance
• Redid MoClo reactions and transformations for all parts
• Ordered promoters for pBad, pTet, pA1LacO, and R0051
• Used colony PCR to verify transformations worked

July

Week of July 7

• Ran gel for colony PCR reactions
• Picked colonies to grow overnight for the parts that turned out well on the gel
  1. BM3RI, BetI, SrpR, LmrA, ECK120015170
• Redid transformations for Ph1F, L3S2P21, SrpR, and ECK120015170
  1. SrpR and ECK120015170 looked questionable on the gel so I redid them just in case
• Miniprepped overnight cultures and sent for sequencing
• Analyzed sequencing results and confirmed BM3R1, BetI, LmrA, and ECK120015170
  1. SrpR came back as LacZ, which means it wasn't properly transformed
• Picked confirmed colonies from stab plate, grew overnight, and made frozen stocks
• Miniprepped SrpR, Ph1F, and L3S2P21 and sent for sequencing
• Analyzed sequencing results and confirmed SrpR and L3S2P21
• Redid colony PCR for Ph1F because it had too low concentration for sequencing

• Diluted pBad, pTet, pA1LacO, and R0051 primers
• PCR for pBad, pTet, and pA1LacO
  1. Held off temporarily on R0051 because we didn't have the R0051_Rev_B primer that we thought we had
  2. For each part we made AK and KB fusion site using the new fusion site, K, that we designed (ATGC)
• Level 0 MoClo reactions in DVL0_AB
  1. pTet-pBad, pTet-pA1LacO, pBad-pTet, pBad-pA1LacO, pA1LacO-pBad, pA1LacO-pTet
• Transformations for level 0 tandem promoter MoClo reactions in bioline cells

Week of July 14

• Created stab plate and picked two colonies from each transformation plate
• Miniprepped overnight cultures and sent level 0 tandem promoter MoClo parts in for sequencing
• Analyzed sequences
  1. Only the pTet-pBad tandem promoter turned out correctly
  2. Noticed that the pA1LacO promoter has a large repeating sequence
     1. PCR temperature that I used was too high (too specific causing reverse primer to bind to the wrong part)
• Redid PCR for pBad (AK) and pA1LacO (AK, KB)

• Received and diluted R0051_Rev_B primer

• Made frozen stocks from the confirmed colonies