Emily

Today I made overnight cultures of my glycerol stock restreaks of I00500, R0040 and B0015 from last week. Tomorrow I will isolate plasmid of these.

Himika

Today I did a plasmid switch for E0040 (GFP). I plasmid switched E0040 from pSB1A2 to pSB1AC3. I did this plasmid switch because B0034 and E0040 are both in A plasmids. I wish to construct a circuit with B0034-E0040 which would not give me a selection pressure. After transformation of E0040 the future construction will give me selection pressure in order to select for the construct.

Procedure:

• Both E0040 and pSB1AC3 was cut with EcoRI and PstI in Invitrogen ReactII Buffer
• Insert E0040
  • 3.5 uL buffer II
  • 0.5 uL EcoRI and 0.5uL PstI
  • 6.0 uL DNA with [102.2 ng/uL]
  • 24.5 uL water

• Vector
  • 3.5 uL buffer II
  • 0.5 uL of EcoRI and 0.5uL PstI
  • 2 uL DNA with [180.0 ng/uL]
  • 28.5 uL water

The restriction digest was allowed to sit in the 37°C hot water bath for 1 hour. Then 5uL of the vector and insert were put in a new tube and 10 uL of quick ligase buffer was added along with 1uL of buffer. This solution was allowed to sit in room temperature for 10 minutes and then they were transformed in Top10 competent cells and plated on chloramphenicol plates.

I also made overnight cultures for I03504 and I03507 in Amp broth.

Dev, Jeremy, Chris

Today, Jeremy did a construction of I0500 (arabinose inducible promoter) to B0034 (Ribosome binding site. He also began the design of our T-shirt logo with Dev as well as finding a colour scheme for the wiki with Patrick. He drew out two possible logos that could be used. In addition, he found a site where customizable T-shirts could be ordered from and experimented with different colour schemes on this. Dev did a construction of E0040 (Green fluorescent protein) to B0015 (double terminators) as well as helping with the T-shirt design and modifying a logo that Himika drew up in the past. Chris did a plasmid switch of B0034 from a kan plasmid to an amp-kan plasmid. He also drew up a possible budget and laid out the possible funding opportunities. For tomorrow, all constructions require a colony PCR as well as overnight cultures made for plasmid preparations.

Patrick

Unfortunately, the restriction digest experiment as outlined yesterday suggests that the cpxP promoters may not have even ligated to the plasmids. Thus, I will retry the insertion of the cpxP into a pSB1AK3 plasmid, which is a plasmid that is compatible with the BioBrick PCR primers that we have available in our lab. These primers are not compatible with pSB1A3, as we have only recently discovered. Thus, I constructed the cpxP promoter into a pSB1AK3 plasmid and transformed it.

As well, I also started learning a bit about the SimBiology toolkit in MATLAB, by MathWorks.

Alex

Another restriction digest/ gel was done on cpxp with Xba and Spe. This time we also used eco and pst to see if the restriction enzymes were indeed cutting the gene out of the plasmid. The results were strange as there ddin’t appear to be any difference between the digested plasmid and the control., leading us to believe we should create another primer so that we can construct cpxp into plasmid directionally. Also, cpxp PCR product was constructed into AK plasmid to attempt to create a successful cpxp biobrick.