Team:Groningen/Parts/Used Parts

From 2009.igem.org

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Used Parts

  • To get to the partsregistry site of a particular part one can click on the name of the part
  • To see what our main findings where (no details or derivations) regarding a particular part one can click on more information

Miscellaneous

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RBS ((Elowitz 1999) -- defines RBS efficiency) 

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iGEM Groningen 2009

The ligation of part Content-type: text/html

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behind the RBS was successful, confirmed by gel (correct vector size after digestion with EcoRI and PstI) and sequencing with VF2 primer. We used this part in combination with several biobricks for building our constructs e.g. Content-type: text/html

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.

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Double terminator (B0012-B0011) 

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iGEM Groningen 2009

The plasmid containing part Content-type: text/html

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was successfully transformed into E. coli TOP10 cells (confirmed by single and double digestion). The original plan was to use the terminator behind one of our own designed parts Content-type: text/html

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, and in front of pArsR-RFP. The MBP-ArsR fusion protein was thought to have a regulating effect on the arsenic promotor pArsR in the same way as ArsR regulates the promotor. The terminator separated the two parts on the plasmid. The construct was not used due to time constraints, and not sequenced.

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GVP Gas Vesicle Proteins 

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iGEM Groningen 2009

Part Content-type: text/html

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was one of our main focus points during our project. The part was originally submitted by Melbourne in 2007 with a very limited amount of information (short description and sequence mutations). On their site it was mentioned the cluster was difficult to use and ligation into a plasmid was hard, in contrast we found the cluster to be easily cut and ligated into different plasmids and behind several promotors. We used this part in combination with several biobricks for building our constructs e.g. Content-type: text/html

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and Content-type: text/html

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. Pictures of our cells with induced gas vesicle formation confirmed the production of vesicles in the expected shape and size. In addition, we characterized the part to improve the use in the future and added a lot of information on the registry.

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ccdB cell death gene

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iGEM Groningen 2009

P1010 is used when putting BioBrick parts into BioBrick plasmids. The part to be inserted and the plasmid are cut with BioBrick enzymes and mixed. The mixture will include both the original uncut or self ligated plasmid and the desired structure. However, because of CcdB, all of the cells containing the original plasmid die and the surviving colonies are the desired result. The ccdB cell death gene worked as expected killing our E. coli TOP10 cells, and keeping our E. coli DB3 cells alive. After noticing the inconsistent sequencing result for the pSB2K3 plasmid with ccdB cell death gene, we decided to choose a different pSB2K3 plasmid with random part to continue with our assemblies. This to minimize the chance of unwanted surprises in the end.

Promotors

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Constitutive promoter family member (high expression) 

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iGEM Groningen 2009

We used a number of the constitutive promoter family members for testing our biobricks. The constitutive promoters show the expected level of fluorescence when transformed into E. coli TOP10 cells. Placing parts behind the promoters turned out to be relatively straight forward. We used this part in combination with several biobricks for building our constructs e.g. Content-type: text/html

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was placed behind the promoters.

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Constitutive promoter family member (high expression, reference) 

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iGEM Groningen 2009

We used a number of the constitutive promoter family members for testing our biobricks. The constitutive promoters show the expected level of fluorescence when transformed into E. coli TOP10 cells. Placing parts behind the promoters turned out to be relatively straight forward. We used this part in combination with several biobricks for building our constructs e.g. Content-type: text/html

Software error:

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was placed behind the promoters.

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Constitutive promoter family member (medium expression) 

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iGEM Groningen 2009

We used a number of the constitutive promoter family members for testing our biobricks. The constitutive promoters show the expected level of fluorescence when transformed into E. coli TOP10 cells. Placing parts behind the promoters turned out to be relatively straight forward. We used this part in combination with several biobricks for building our constructs e.g. Content-type: text/html

Software error:

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was placed behind the promoters.

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Constitutive promoter family member (low expression) 

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iGEM Groningen 2009

We used a number of the constitutive promoter family members for testing our biobricks. The constitutive promoters show the expected level of fluorescence when transformed into E. coli TOP10 cells. Placing parts behind the promoters turned out to be relatively straight forward. We used this part in combination with several biobricks for building our constructs e.g. Content-type: text/html

Software error:

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and Content-type: text/html

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were placed behind the promoters.

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Promoter (lacI regulated) 
This part is an inverting regulator sensitive to LacI and CAP. It contains two protein binding sites. The first binds the CAP protein, which is generally present in E.coli and is asocciated with cell health and availability of glucose. The second binds LacI protein.

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pBad/araC 

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iGEM Groningen 2009

The sequence was listed as inconsistent, and the ligations of parts behind the promoter failed. Restriction of isolated plasmids showed fragments of unexpected sizes.

Vectors

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High copy BioBrick assembly plasmid

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iGEM Groningen 2009

pSB1AC3 is a high copy number plasmid carrying ampicillin and chloramphenicol resistance. Together with pSB1A2 the vector was used for most assemblies of our team, because the high copy number made it easy to work with and easy isolation. The transformations with pSB1AC3 (containing different biobricks of own design) into E. coli TOP10 cells, growth on both antibiotics, and gel analysis (undigested and digested with the EcoRI and PstI) worked as expected. The high (copy) number of plasmids per cell make it an easy to work with plasmid, ideal for cloning and assembly work.

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pSB1A2 (Replaced by pSB1A3 in registry)  
pSB1A2 is a high copy number plasmid carrying ampicillin resistance. Together with pSB1AC3 the vector was used for most assemblies of our team, because the high copy number made it easy to work with and easy isolation.

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A low copy number base vector 
A low copy number base vector which as a resistance against Kanamycin

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A low copy number base vector 
A low copy number base vector which as a resistance against Kanamycin

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A normal base vector 
A normal base vector which has a resistance against Ampicillin

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Vector we get with GVP

It's the backbone of our GVP part is and it a resistance against Ampicillin/Gentamycin