Team:UNIPV-Pavia/Calendar/August/settimana4

From 2010.igem.org


AUGUST: WEEK 4



August, 23rd

Colony PCR as screening for ligations I31..I41. For each plate three colonies were picked and PCR amplification was performed. At the same time colonies were let grow in 750 ml LB+Amp in order to be ready to make glycerol stocks for those with right amplicons.

Colony PCR and gel run as screening for ligations I31..I41

Only I31, I33, I34, I35, I37, I38, I41 are positive without any doubt (we took I31-1, I33-1. I34-1, I35-1, I37-1, I38-3, I41-1), while I39 were all wrong and we weren't sure about I32, I36 and I40 (we made however glycerol stocks for I32-2, I36-2, I40-1). We decided to repeat colony PCR for I32, I36, I39 and I40 next day.


MyCrim-9, BBa_K173001, BBa_J23101, pSB4C5 were digested E-P and MyCrim-9, Ring were digested HindIII for three hours.

Culture Kind Final reaction volume (ul) DNA (ul) H20 (ul) Enzyme 1 (ul) Enzyme 2 (ul) Buffer (ul)
MyCrim Vector 25 6,5 14 1 EcoRI 1 PstI 2,5 H
MyCrim Vector/Screening 25 4 16,5 1 HindIII 1 HindIII 2,5 B
BBa_K173001 Insert 25 8 12,5 1 EcoRI 1 PstI 2,5 H
BBa_J23101 Insert 25 5 15,5 1 EcoRI 1 PstI 2,5 H
pSB4C5 Insert 25 5 15,5 1 EcoRI 1 PstI 2,5 H
Ring Vector/Screening 25 4 16,5 1 HindIII 1 HindIII 2,5 B

Gel extraction of BBa_I52002(E-P) (insert of pSB4C5), MyCrim (E-P), BBa_K173001 (E-P), MyCrim (HindIII), vector of pSB4C5. Quantification:

Sample Quantifications"
MyCrim (HindIII) 13 ng/ul
MyCrim (E-P) 23,4 ng/ul
BBa_K173001 (E-P) 11 ng/ul
BBa_J23101 (E-P) 9 ng/ul
pSB4C5 vector (E-P) 23 ng/ul
BBa_I52002 (insert of pSB4C5) (E-P) 6 ng/ul

These parts were used to perform following ligations:

Name Vector Insert
I42 MyCrim (E-P) BBa_K173001 (E-P)
I43 MyCrim (E-P) BBa_J23101 (E-P)
I44 MyCrim (E-P) BBa_I52002 (insert of pSB4C5) (E-P)
I45 MyCrim (HindIII) MyCrim (HindIII)

August, 24th

Only this morning we remembered that I40 is in a commercial vector (pMA), we will screen it next day through an E-P digest (today we made glycerol stocks for other four colonies: I40-4/5/6/7) since it's impossible through colony PCR.

With this method we checked four new colonies of I32 (I32-4/5/6/7), I36 (I36-4/5/6/7) and I39 (I39/4/5/6/7).

Colony PCR and gel run as screening for ligations I32, I36, I39

I39-4/5 are right (we made glycerol stock for I39-5); I32 and I36 still show the presence of cotrasformation of our ligation and a simple phasin (we took I36-7 instead of I36-2: cleaner gel run). We inoculated 5ml LB+Amp with I40-1/4/5/6/7 and we let them grow ON at 37°C, 220 rpm for E-P digestion/screening.


Inoculum of:

for tomorrow MiniPrep. They will be processed with other promoters, for wich we retrieved purified DNA from our freezer. These further parts are:

These promoters, expressing RFP, will be moved from high copy plasmid pSB1A2 to the low copy plasmid pSB4C5 and will be tested in both condition in order to establish a strength ranking.


Inoculum of PBHR68 BioPlastic producing device and BBa_B0032 from glycerol stock in 5ml LB+Amp to check the production of BioPlasic with Sudan Black staining protocol.

Cultures were grown ON at 37°C, 220 rpm.


I42, I43 ligations were transformed in BW23474 strain and plated on selective LB agar plate with chloramphenicol concentration of 34 ug/ml. Instead I44 was transformed in DB3.1 strain and plated on the same type of plate of the other two transformations.


August, 25th

Minipreps of I40-1/4/5/6/7 (to make the screening of ligations) were quantified as follows:

  • I40-1: 415,7 ng/ul
  • I40-4: 382,8 ng/ul
  • I40-5: 420 ng/ul
  • I40-6: 487,8 ng/ul
  • I40-7: 479,9 ng/ul

Samples were digested E-P for three hours

Culture Kind Final reaction volume (ul) DNA (ul) H20 (ul) Enzyme 1 (ul) Enzyme 2 (ul) Buffer H (ul)
I40-1 Insert/Screening 25 2 19,5 0,5 EcoRI 0,5 PstI 2,5
I40-4 Insert/Screening 25 2 19,5 0,5 EcoRI 0,5 PstI 2,5
I40-5 Insert/Screening 25 2 19,5 0,5 EcoRI 0,5 PstI 2,5
I40-6 Insert/Screening 25 2 19,5 0,5 EcoRI 0,5 PstI 2,5
I40-7 Insert/Screening 25 2 19,5 0,5 EcoRI 0,5 PstI 2,5

and than gel run to check the length of ligations.

Gel run for I40-1/4/5/6/7 digested E-P

As you can see all samples are positive, so we decided to keep glycerol stock of I40-1.


MiniPrep was performed for following cultures, and DNA was quantified as follows:

Culture name
BBa_J23105
BBa_J23106
BBa_J23114
BBa_J23116

Purified DNA was digested as follows:


Digestion of:

Culture Kind Final reaction volume (ul) DNA (ul) H20 (ul) Enzyme 1 Enzyme 2 Buffer H
BBa_J23100 Insert 25 10 10,5 1 EcoRI 1 PstI 2,5
BBa_J23105 Insert 25 10 10,5 1 EcoRI 1 PstI 2,5
BBa_J23106 Insert 25 10 10,5 1 EcoRI 1 PstI 2,5
BBa_J23110 Insert 25 10 10,5 1 EcoRI 1 PstI 2,5
BBa_J23114 Insert 25 10 10,5 1 EcoRI 1 PstI 2,5
BBa_J23116 Insert 25 10 10,5 1 EcoRI 1 PstI 2,5
BBa_J23118 Insert 25 10 10,5 1 EcoRI 1 PstI 2,5

Ligation of:



Preparation of samples for BioPlastic screening:

  • Cultures of PBHR68 and BBa_B0032 were diluted 1:100 in fresh LB+Amp and werew prepared as follows:
    • BBa_B0032 with NOTHING added (negative control)
    • PBHR68 with NOTHING added
    • PBHR68 + 2% glycerol (carbon source for BioPlastic production)
    • PBHR68 + 1mM IPTG (inducer for Plac promoter, expressing BioPlastic enzymes)
    • PBHR68 + 2% glycerol + 1mM IPTG

After 8 hours, Sudan Black staining protocol was performed on 70ul cultures and 5 microscope slides were prepared. The resulting images are shown here:

PBHR68 with nothing added in the culture, after 8 hours
PBHR68 with nothing added in the culture, after 8 hours
BBa_B0032 with nothing added in the culture, after 8 hours (negative control)
BBa_B0032 with nothing added in the culture, after 8 hours (negative control)
PBHR68 with 2% glycerol added in the culture, after 8 hours
PBHR68 with 2% glycerol added in the culture, after 8 hours
PBHR68 with 1mM IPTG added in the culture, after 8 hours
PBHR68 with 1mM IPTG in the culture, after 8 hours
PBHR68 with 1mM IPTG and 2% glycerol added in the culture, after 8 hours
PBHR68 with 1mM IPTG and 2% glycerol added in the culture, after 8 hours

Cultures were further incubated for 22 hours at 37°C, 220 rpm. Tomorrow, we will repeat the staining protocol after 30 hours from inoculum to check the time E. coli takes to produce BioPlastic granules.

August, 26th

Transformation of ligations:

Ligation name Strain Resistance
BBa_J23100_4C5 TOP10 Cm 12,5
BBa_J23101_4C5 TOP10 Cm 12,5
BBa_J23105_4C5 TOP10 Cm 12,5
BBa_J23106_4C5 TOP10 Cm 12,5
BBa_J23110_4C5 TOP10 Cm 12,5
BBa_J23114_4C5 TOP10 Cm 12,5
BBa_J23116_4C5 TOP10 Cm 12,5
BBa_J23118_4C5 TOP10 Cm 12,5

Plates were incubated at 37°C overnight.

Inoculum of self inducible promoters for TECAN test:

I7 I8 I9 I10
I12 I8_4C5 I15 I16
I17 I18 I19 I27
I28 I30 BBa_B0032 A2 (BBa_J23101+GFP)

After 30 hours, Sudan Black staining protocol was performed on 70ul cultures and 5 microscope slides were prepared. The resulting images are shown here:

PBHR68 with nothing added in the culture, after 30 hours
PBHR68 with nothing added in the culture, after 30 hours
BBa_B0032 with nothing added in the culture, after 30 hours (negative control)
BBa_B0032 with nothing added in the culture, after 30 hours (negative control)
PBHR68 with 2% glycerol added in the culture, after 30 hours
PBHR68 with 2% glycerol added in the culture, after 30 hours
PBHR68 with 1mM IPTG added in the culture, after 30 hours
PBHR68 with 1mM IPTG in the culture, after 30 hours
PBHR68 with 1mM IPTG and 2% glycerol added in the culture, after 30 hours
PBHR68 with 1mM IPTG and 2% glycerol added in the culture, after 30 hours

Inoculum of

in 5 ml LB+Amp

August, 27th

Previously inoculated strains were miniprepped and quantified as follows:

  • BBa_J13002: 29,9 ng/ul
  • I33: 171,6 ng/ul
  • I34: 219,4 ng/ul
  • I39: 21,2 ng/ul
  • I41: 94,7 ng/ul

Digestions (3,5 hours) for ligations:

  • I47: BBa_J13002 (S-P) + I33 (X-P)
  • I48: I39 (S-P) + I34 (X-P)
  • I49: I39 (S-P) + I41 (X-P)
Culture Kind Final reaction volume (ul) DNA (ul) H20 (ul) Enzyme 1 (ul) Enzyme 2 (ul) Buffer H (ul)
BBa_J13002 Vector 25 21,5 0 0,5 SpeI 0,5 PstI 2,5
I39 Vector 25 21,5 0 0,5 S 0,5 P 2,5
I33 Insert 25 10,5 11 0,5 XbaI 0,5 P 2,5
I34 Insert 25 13,5 8 0,5 X 0,5 P 2,5
I41 Insert 25 2 19,5 0,5 X 0,5 P 2,5

Samples were loaded into a medium gel, run and cut

Samples gel run/cut

Gel extraction was quantified as follows:

  • BBa_J13002 (S-P): 19,9 ng/ul
  • I33 (X-P): 17,7 ng/ul
  • I34 (X-P): 11,1 ng/ul
  • I39 (S-P): 4,7 ng/ul
  • I41 (X-P): 6,2 ng/ul

Ligation of

  • I47: BBa_J13002 (S-P) + I33 (X-P)
  • I48: I39 (S-P) + I34 (X-P)
  • I49: I39 (S-P) + I41 (X-P)



Plates containing the transformed ligations were all grown. Colony PCR was performed to screen if the length of insert was correct.

ligation name number of colonies screened
BBa_J23100_4C5 2 colonies
BBa_J23101_4C5 2 colonies
BBa_J23105_4C5 2 colonies
BBa_J23106_4C5 2 colonies
BBa_J23110_4C5 2 colonies
BBa_J23114_4C5 1 colony
BBa_J23116_4C5 2 colonies
BBa_J23118_4C5 2 colonies


PCR screening for Anderson Promoters transferred in low copy plasmid pSB4C5

Gel results show that we have the correct clones for BBa_J23100_4C5, BBa_J23101_4C5, BBa_J23105_4C5, BBa_J23106_4C5, BBa_J23110_4C5, BBa_J23114_4C5. Glycerol stocks were prepared for these parts and are stored at -80°C.

BBa_J23116_4C5 and BBa_J23118_4C5 need to be further screened, so we will pick other colonies next week and we will perform a new colony PCR.


Tecan Test was performed on prepared samples, after the usual protocol (dilution, medium change and dilution 1:1000).


Transformation of I29 in E. coli TOP10

August, 28th

Ligations I47, I48, I49 were stored at +4°C.


I29 plate was stored at +4°C.