Team:Uppsala-Sweden/Notebook

Week 1

This week was dedicated to make buffers such as CCMB80 and SOC, SOB and LB medium (See protocol SOB- medium, LB medium and Competent cell preparation final). We also prepared selective agar plates with ampicillin, chloramphenicol and kanamycin (See agarplate preparation). Finally we prepared competent TOP10 cells and froze them in -80°C (See Competent cell preparation final).

Week 2

2011-06-27

• This week we started with testing the competence in our cells. For this transformation we used one positive control (plasmid pUC19 from New England Biolabs) as well as a negative control without plasmid. For the procedure we followed the protocol for transforming TOP10 competent cells. The result was good, we measured a competence efficiency of 1.7 * 10^8 transformants /ug DNA.

• Started overnight cultures of E coli carrying the plasmids pGEM11- amilGFP (green output) and pGEM14- amilCP (blue output). For this procedure we followed the protocol Overnight culture and glycerol stock. The strains carrying the amilGFP and amilCP plasmids were provided by J.F Miller, UCLA.

2011-06-28

Transformation of BioBrick plasmids (protocol for transforming TOP10 competent cells):

pSB1A3-J04450 (ampR backbone) pSB1C3-J04450 (CmR backbone) pSB1K3-J04450 (KanR backbone) pSB1AK3-B0014 (Double terminator) pSB1AK3-B1001 (synthetic terminator) pSB1A2-B0034 (Standard RBS) pSB2K3-I15008 (ho1, chromophore gene) pSB2K3-I15009 (pcyA, chomophore) pSB1A2-R0011 (PllacO, lacI repressable promotor) pSB2K3-I15010 (cph8 red sensor)

2011-06-29

Transformation of BioBrick plasmids (protocol for transforming TOP10 competent cells):

pSB2K3-Q03530 (cII inverter) pSB3T5-J04450 (low copy vector tetR, ori P15A) pSB1AK3-B0015 (Double terminator) pSB2K3-Q01511 (cI inverter) pSB1A2-R0082 (PompC) pSB1A2-K093005 (RBS + RFP, red dye) pSB2K3-I15010 (cph8 red sensor) - had to be redone due to failure in the first attempt.

Followed up by restreaking of the transformants from the previous day.

2011-06-30

Restreaking of the transformants from 11-06-29 and started overnight cultures of the reastreaked plates from the previous day (11-06-28).

2011-07-01

Started with doing overnight cultures from the reastreaked plates from the previous day (11-06-30). After that we prepared 20 % sterile glycerol for making glycerol stocks. All the overnight cultures prepared 11-06-30 were frozen in -80°C.

2011-07-02

All the overnight cultures prepared on the previous day were made into glycerol stock and frozen in -80°C.

Week 3

2011-07-04

Started by doing overnight cultures of the strains carrying these plasmids:

pSB1A3-J04450 (vector, ampR) pSB1C3-J04450 (vector, CmR) pSB1K3-J04450 (vector, KanR) pSB1A2-B0034 (Standard RBS) pSB2K3-I15008 (ho1, chromophore gene) pSB2K3-I15009 (pcyA, chomophore) pSB2K3-Q03530 (cII inverter) pSB1AK3-B0015 (Double terminator) pSB2K3-Q01511 (cI inverter) pSB1A2-R0082 (PompC) pSB2K3-I15010 (cph8 red sensor)

The purpose was to make plasmid preparation the day after.

2011-07-05

The day started of by running a PCR on the DNA template of the green light sensor which includes the two genes ccaR and ccaS as well as the promotor PcpcG2 (see protocol ccaR BioBrick, protocol ccaS BioBrick, protocol PcpcG2 BioBrick).

After lunch we did a plasmid preparation of the overnight cultures from 4/7 (purification protocol). At the end of the day we did a PCR to verify the red light sensor (cph8). We also did site specific mutagensis using PCR for the pigments amilGFP and amilCP in order to remove illegal EcoRI sites within the genes (protocol cph8, protocol amilGFP_EcoRI, protocol amilCP_EcoRI)

2011-07-06

Preparation of agarose gel (1%) for the gel electrophoresis. Then we ran our PCR products from yesterday (11-07-05) in the gel electrophoresis. Finally we initiated the first biobrick assembly session. The following entities were assembled pcyA-RSB, ho1- RSB and Inv.cl-PompC, ho1-gene and RBS.

2011-07-07

re-circularized of pigment vectors after site mutation PCR (Purification protocol, Phosphorylation of DNA, DpnI digestion protocol). Transformation of the ligated plasmids from 11-07-06 (Protocol for transforming TOP10 competent cells). Transformation of mutagenized amilCP and amilGFP. Digestion of ccaS, ccaR and plasmid backbone plasmid pSB1C3 (BioBrick Assembly Manual).

2011-07-08

PCR of EnvZ- Knock out FRT-Kan, Cph8 and pcpcG2 (PCR protocol of envZ knockout FRT-Kan_FRT,...). Transformation of Chp8. Re streak of the transformants from 2011-07-07.

2011-07-10

Today we did Screening of transformants from 11-07-08 (3A assemblies: pcyA-RSB, ho1- RSB and Inv.cl-PompC, ho1-gene and RBS, ccaS and ccaR, amilCP and amilGFP, 6 clones of each, 42 samples in total). We Picked and suspend a colony from each re-streak in 20-30 µl of PBS. The volume depends on the colony size. Small colonies use 15-20 µl, big colonies take 30 µl. We also started overnight culture in selective medium from the suspensions, 1 ml in each. Run colony PCR from of each suspended colony using taq polymerase. Last thing was run an agarose gel of PCR products.

Week 4

2011-07-11

Today we did a frozen stock of all viable stocks in -80°C freezer (ccaS clone 1,RBS-pcyA, amilGFP, amilCP, ccaR, RBS-ho1, PompC-Inv.cI). We also did a 3ml culture of the two clones of ccaR, RBS-pcyA and RBS-ho1, the constructs needed for the upcoming assemblies. Furthermore, we did a plasmid preparation of this clones. Transformation of tetR and lambda CI from MIT distribution. PCR of amilCP to add BioBrick prefix and suffix. Mutagensis of amilGFP-PstI and ccaS mutagenesis, round one.

2011-07-12

The day started by running a gel of ccaS mutant, amilCP-BB10, and amilGFP-PstI. At the same time we prepared the sample (ccaR, ccaS, RBS-ho1 and RBS-pcyA) for sequencing. Plating of terminator B1001, PLlacO, pSB3T5 and pSB4K5. PCR purification of ccaS, amilGFP, amilCP, clone amilCP and PcpcG2 (Purification protocol). Re-ligation and transform mutagenized ccaS and amilGFP. Transformation of amilCP, clone amilCP, TetR inverter and pcpcG2. Re-streak of cI inverter transformants from 2011-07-11.

The transformation of inverters from yesterday didn’t go well. The lambda-cI-inverter had only a few colonies, while the tetR inverter had no colonies at all. So TetR inverter has to be redone. This time the transformation used 2 µl of DNA from the MIT distribution.

Furthermore new plates containing all antibiotics and some containing teracyclin were made.

2011-07-13

We started the day by running EnvZ-KO, ccaS, EcoRI- digested amilCP and amilGFP on a gel. We obtained good result since there were no distinguishable multiple bands in the gel, although the “single band” seemed a bit smeared. Then we did transformation of ccaS_EcoRI, amilGFP_PstI, pSB1A3-amilCP, pSB-pcpcG2 and pSB4A5 backbone. Re-streak (There were one colony on the whole plate) and PCR of tetR inverter (DNA taken from well distributed from MIT).

o/n of B1001 terminator, PlacO, PSB3T5 and PSB4K5-backbone (prepared for frozen stock + plasmid preparation) and finally RFP with RBS and lambda c1.inv (prepared for frozen stock + plasmid preparation). The last thing we did was to make new plates of all the three antibiotics (Am,k, Ch). We also made some plate with Tetracyclin

2011-07-14

Re-streak pSB1C3-ccaS_EcoRI, pGEM-amilGFP and pSB4A5. We were supposed to re-streak pSB1A3-amilCP and pSB1A3-PcpcG2 as well, but the transformants had no colonies at all. Suspect wrong plasmid backbone or no ligase added during the cloning attempt. To investigate this we did a transformation of the plasmid backbone (PSB1A3) and plated it on plates containing antibiotics ampicillin, chloramphenicol and kanamycin. We did plasmid prep of pSB1AK3-B1001, pSB1A2-R0011, pSB3T5-backbone, pSB4K5 backbone. New agar plates with antibiotics ampicillin, chloramphenicol and kanamycin, just made in the morning. Assembly of chromophores, step Cloning of tetR inverter into new backbone. Cloning of pSB1A3-amilCP and pSB1A3-PcpcG2 again. Overnight culture of pSB1A3 from the strain collection to test whether it’s right.

The sequencing result arrived today around lunch time! RBS-pcyA strains we sent for sequencing were good. ccaR clone number 2 is better than clone number 1. Clone 1 of RBS-ho1 will be used in further assembly steps.