Prepared for the advisor meeting today:
Pgad promoter update from the CUHK team-
The Pgad part will be shipped in a plasmid and we will have to PCR it out

Website update-
Yuan is out for the week so website progress is halted; however, the protocols and lab notebook should be online by the end of the week

Human practices update-
Lajja is talking to the CTD directors about possibly teaching a class on synthetic biology.
Mike is continuing to contact government officials to get a meeting to discuss synthetic biology.
Brian is continuing to work with the Cornell group on the group of synthetic biology videos.

The lab worked on transforming the lysis part from Berkeley 2008 into e. coli

K12 DNA: 314.8 ng/uL 1.90 absorbance ratio

Lajja heard back from the CTD administrators and they would be happy to have us visit a biology class.
Grant worked on preparing a presentation for the CTD kids; however, their age is still unknown so the level of the material can vary
The primers for e. coli phytase and e. coli clc antiporter came in!
PCR was performed to get the ECphy and the ECClc out of the K12 e. coli genome

Lajja confirmed that the age of the CTD kids will be 15-17 and that they already know much about biology
Grant worked on preparing a more accurate presentation for 15-17 year old CTD kids.
The team brainstormed possible avenues of keeping older kids interested in a presentation. Specifically, ideas about transforming some simple biobricks from the registry that did “cool” things were thrown around. For example: GFP/RFP/CFP, luciferase, e. coli that smell like bananas or wintergreen
Brian, Grant, and Mike went to a biotechnology lecture on modeling in metaboloic engineering. Although insightful, much of the lecture was above our heads.
Sarah and Grant performed a gel on the PCR products from yesterday (ECphy and ECClc)
The gel results can be seen here: [LINK]
The gel verifies that the two PCR’d parts are:
e. coli phytase: 1299 nucleotides
e. coli clc antiporter: 1564 nucleotides

Sarah and Grant performed a PCR purification using Qiagen’s PCR purification kit; however, the resulting DNA was of low concentration(20 ng/ul for ECphy and 25 ng/ul for ECClc)
Prof. Mordaq suggested to continue with digesting the DNA despite the low concentrations of DNA. However, he also suggested to do another round of PCR just in case.

The PCR products from last night’s redo of ECPhy and ECClc was ran on a gel
The PCR products had small bands of nucleotides and primers, but were otherwise at the correct position on the gel
The digested products from the original PCR were isolated well and were pure. The bands lined up at the correct nucleotide positions
We ligated the ECPhy and the ECClc into the pSB1A3 backbone
Brian transformed CFP, the pSBA plasmid backbone, and Pbad from the registry. CFP was transformed for use in the CTD demonstration next week. The pSBA plasmid backbone was transformed for miniprepping and eventually making more linearized plasmid backbones. The Pbad promoter was transformed to test the lysis part in an inducible way (arabinose turns on lysis part).

The ECPhy and ECClc in the pSB1A3 backbone were transformed into e. coli

Overnights of bacillus subtilus and citrobacter braaki were made following a modified version of the usual overnight protocol:

1. Take off aluminum and rubber stopper
2. Add .5 mL LB to the vial of bacteria
3. Add 5mL LB to the overnight tube
4. Mix the LB and bacteria in the vial
5. Add the bacteria to the LB in overnight
6. Leave overnight

Overnights of CFP, the plasmid backbone, and Pbad were made.

100 uL of the citrobacter braaki and bacillus subtilus bacteria were plated on LB media
CFP, the plasmid backbone, and Pbad were miniprepped.