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My Construction Files
My Sequencing Files

Dtulga - May 30th - June 5: Group Work

Did the first cloning of the part Bca1145 from Bca1144. This involved first PCR'ing out the modified RFP gene from the p9145-Bca1144#5, then digestion and purification of the vector p9145.

We then transformed some DH10 cells with the new part, and cutured 2 clones. We then miniprep'd them, as well as did digestion and colony PCR. Our colony PCR was unsuccessful, but the miniprep digestion indicated our part was correct.

We then sequenced the data and found that both sequencing files appear correct, although there was a possible sequencing error of calling an incorrect number of repeating AAA's in clone #1.

Sequencing Files: AY001, AY002

Dtulga - June 4-5: Independent Work

Designed construction files for pI716101 (Low Copy ampR Plasmid - Produces RFP), pir I716102 (R6K Origin Inducer), and T7rnap I716103 and I716104 (T7 RNA Polymerase).

This includes the first few genes for the controller, including the low copy controller plasmid that will control the inducible BAC.

I also transformed some TG1 cells with the plasmid pSB4A3-I7101, for a midiprep of the low-copy plasmid we'll use for pI716101. I also cultured them in 100ml of Amp LB.

Basic Part Construction Progress

Progress Status

Designing : Making Construction Files and Ordering Oligos
Building : PCR, Transforming, Culturing
Checking : Analytical Digesting and Sequencing
Confirmed : Correct clones are confirmed and mini/midiprep'd
Finished : Correct clones are organized and saved

Low Copy ampR plasmid (pI716101) Construction - Confirmed

The midiprep of the plasmid pSB4A3-I7101 was successful, and I additionally digested and ligated the new 1144 Fragment into its EcoRI/XhoI insert to convert it into the Biobricks 2.0 Format.

The colonies grown from this ligation/transformation were both red and white - the red ones being the Bca9145-1144 parent vector, while the white colonies I picked appear to be the correct product.

I then did a test digest with PstI and HindIII, and the results indicated that the red colonies were parent, and the large white colonies were the correct product. Additionally, upon pelletization, the red colonies' cells appear distinctly red, while the white colonies appear slightly tinted red, which is the exact phenotype expected for the product, a low-copy plasmid with a RFP producing device.

June 19 to June 21: A midiprep of clone #3 (large white colony) was then done, and the plasmid DNA was run, as well as a mapping digest, with 4 different enzyme pairs, was performed. The plasmid DNA gel matched the expected size perfectly, and the mapping digest appears correct. I can eliminate the possibility of original biobricks 1.0 vector, as the product is cut with BglII, which was not present in the original vector. As well, the phenotype and size/map digest strongly support the indication that the product is correct. Therefore, clone #3 is confirmed to be correct, and likely clone 4 is additionally right, but has not been extensively tested.

pir (pI716102) Construction - Building (Growing Re-Do Transformation)

I attempted many PCR reactions for pir, many of which failed, or gave too dilute a product to transform with. I additionally found that some of the incorrect colonies which were grown up from the first PCR product that appeared to succeed were actually primer dimers, and some were also mixed clones.

June 21: I have re-tried PCR from the overlap A+B step, as both the A and B products did succeed. The resulting transformation from that PCR has been grown up on an AMP plate.

T7 RNA Polymerase ATG and GTG starts (pI716103, I716104) Construction - Building (Re-Do PCR)

I have attempted numerous PCR reactions for the T7 RNA Polymerases, and so far none have succeeded, either by returning no product, incorrect product due to mis-annealing, or genomic bleed through.

I will attempt the PCR reactions again, potentially from a different template, when we receive more Expand polymerase.

T7 Promoter Variants (I716105, I716106, I716107) Construction - All Confirmed

I designed and ordered oligos to create the T7 promoters synthetically, as their short length (29bp), and multiple variants (Wild Type, D, and E) mean that creating them from an extension of two annealing primers was the best method.

It succeeded in producing products and colonies for all three variants. Two clones for each were sent off for sequencing, and one of each variant came back perfect. The other three were an unusual RFP parent recombination. Therefore, clones: 1 for I716105, 1 for I716106, and 2 for I716107 are correct.