Team:Goettingen/Parts

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The beast and its Achilles heel:

 A novel target to fight multi-resistant pathogenic bacteria



Parts

Parts we used in our project

Internal Part No.

Registry No.

Function

Strength (au)

Length (bp)

whereabout
(in 2013kit)

Backbone/Resistance

 

Important additional Info

1

BBa_J23117

Promoters

162

35

P5 20 O

BBa_J61002

Amp

 

2

BBa_J23116

396

35

P5 20 M

BBa_J61002

 

3

BBa_J23110

844

35

P5 20 C

BBa_J61002

 

4

BBa_J23118

1429

35

P5 22 A

BBa_J61002

 

5

BBa_J61101

RBS

rather low

 

P5 5 L

pSB1A2

Amp

 

8

BBa_B0034

very strong

12

P5 2 M

pSB1A2

Amp

 

6

BBa_E0240

GFP generator

(with promoter and RBS)

 

876

P3 23 A

pSB1C3

(high copy)

Cm

Excitation: 501 nm

Emission: 511 nm

Latency: 8 min

 

Literature:

Cormack, B.P. et al  (1996)

 

P5 12 M

pSB1A2

(high copy)

Amp

7

BBa_B0015

Terminator

 

129

P3 4 F

pSB1C3

(high copy)

Cm

 

9

BBa_E0030

EYFP (no promoter, no RBS)

 

723

P3 16 D

pSB1C3

(high copy)

Cm

Excitation: 514 nm

Emission: 527 nm

10

BBa_E0020

ECFP (no promoter, no RBS)

 

723

P3 3 M

pSB1C3

(high copy)

Cm

Excitation: 439 nm

Emission: 476 nm


Parts we modified/improved/created


<groupparts>iGEM013 Goettingen</groupparts>


Promoter Characterization

To construct our reporter systems, we needed promoters of different strength. The well-known Anderson promoter collection consists of nineteen constitutive promoters of different strength. Because of their specific strength, we chose four different promoters (Table 1).

Intern No. for promoter

Registry No.

Strenght in arbitrary units

(according to the characterization of the iGEM Team Berkeley 2006)

Promotor 1

BBa_J23117

126

Promoter 2

BBa_J23116

396

Promoter 3

BBa_J23110

844

Promoter 4

BBa_J23118

1429

Table 1. For our project, we were interested in four different promoters from the Anderson Collection. For each part, we used an intern number, which will be used in the text, as well.

The activity of these promoters was mainly characterized by measurement of the activity of a reporter protein expressed from these promoters. Protein activity is not only determined by the efficiency of transcription. In addition to this, translation efficiency contributes to the protein amounts in a cell. In particular, regarding fluorescent proteins like RFP or GFP, the growth phase might affect their fluorescence (see discussion below). A more direct way to determine the promoter activity is to assess the transcript levels. We were interested in how the mRNA levels of RFP placed downstream of the promoters from the Anderson collection change dependent on the promoter and how the protein activity compares to them.

For this, we transformed E. coli DH5α with the promoter parts present in the plasmid backbone BBa_J61002. As an empty vector control lacking a promoter and RFP, we used E. coli cells transformed with BBa_B0034(an RBS) in plasmid Backbone pSB1A2. Subsequently, we characterized three clones of each strain by qRT-PCR and by monitoring the RFP fluorescence in different growth stages. In addition to this, we did fluorescence microscopy of exponentially growing cells (Fig. 1).

For qRT-PCR analysis (Fig. 2), E. coli cells were harvested at an OD600nm of ca. 0.6 (exponential growth phase) and after 5 – 6 h when the OD600nm had reached approximately 2 (stationary phase). The cells were incubated over night and a third sample taken the next morning ( > 20 h incubation, OD600nm was ca. 2 – 3). The relative mRNA levels of rfp were normalized to the rpoA levels. Then, the fold change (∆∆CT) of the promoters compared to promoter 1 (the weakest) was calculated for each phase. The fold enrichments obtained for different growth phases could not be compared since the CT values determined for rpoA suggested a generally altered transcription during the different growth stages.

 

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