Week 6 Lab: Gibson Assembly

cover image

Lab6

For lab this week we conducted a Gibson assembly to change the color of the purple Acropora millepora chromoprotein to a different mutant colors. I worked with Sean Murphy and Terry Luo, and we selected purple, orange, and light pink but ended up with 9 different petridishes to test our whole process, including: 4 x Purified DNA -> Gibson Assembly -> Transformation (but all mixed up and at much too low concentrations, there are four of these because one is all backbone) 3 x DNA straight from PCR -> Gibson Assembly -> Transformation (much higher concentrations but not purified) 1 x A mix of all of our purified DNA to see if we could get the concentration high enough and just see what happened 1 x A test with just the template plasmid A lot went wrong, so we really tried to troubleshoot.

Part 1: PCR

PCR went quite well, we had no problem preparing for PCR and tests of our PCR product on an e-gel confirmed that we were successful. We had to run two gels because we forgot to run our backbone the first time.

e-gel image

e-gel images

When purifying our DNA, we mistakenly disposed of our bound DNA after the wash matrix step. Luckily, we were able to recover it, but suspect putting all of our product into the bio waste container had something to do with our low concentration later on. This step also led to us mixing up our purified DNA such that we could not tell which color was what and morre importantly which tube contained our backbone.

centrifuge image

Centrifuge image

When measuring our eluted purified DNA, we ran into some concentration problems. The nanodrop read the concentration of our elution at 2ug/ml, 4ug/ml, 6ug/ml, and 7ug/ml compared to the template at 40ug/ml. Different templates read different amounts, with the lowest only reading around 12ug/ml so this might have been a problem but more than likely we lost some DNA to the trash, whether from when we threw it out or from problems with eluting.

Nanodrop image

Nanodrop Image: our purified DNA saw no real peak, but our PCR product saw a huge peak with only a little bit of noise

For the Gibson Assembly step, we decided to create 9 different products to try our luck at all possible combinations of purified and unpurified DNA, and because we could not distinguish our purified backbone, we used an unpurified backbone for all of them.

Nanodrop image

This is how they came out:

Purple image

These are the two purples, unpurified on the left and purified on the right

Orange image

These are the two oranges, unpurified on the left and purified on the right

Light Pink image

These are the two light pinks, unpurified on the left and purified on the right

Mix image

Here is the mixed one

Backbone image Control image

And here is what we are assuming is the double backbone to the left, and control to the right Overall, we did have success for each of our plates; however, very few of our colonies were transformed as most of them still appeared purple.