Unit 3: iGEM Common Practices

Obtaining the Parts’ Sequence

The model of composite parts will be demonstrated in unit 3. However, the design of parts can be very complicated and dynamic, in order to serve different desired functions. Therefore, when researching on papers, also notice the design from the paper. Scientist who publish the paper is much more experienced than us and their construct is more likely to work.

Undocumented parts in iGEM

If you are going to use any sequence that has not been documented by iGEM, you need to find the sequence outside iGEM.

They are usually obtained when reading papers. You may want to learn from their project and use the same gene sequence they used. The papers are usually embedded with the gene they found or used. We can directly copy from the paper without worrying about copyright or patent issues. If the sequence is used instead of discovered in that research, it is likely that the sequence is not directly attached to the paper, you may use ResearchGate (www.researchgate.net) to find the sequence used in the research. The used sequence is usually attached at the end of the paper as supplementary resources. You may download the file then open it with SnapGene, then copy it for further use.

1. Search for the research publishment

2. Scroll until you see ‘supplementary resources’

3. Click into the desired sequence

4. Download .gb file

5. Open file with SnapGene

If you find the sequence of a certain known gene, NCBI comes in handy.

Search for the gene name in the category of nucleotides in NCBI

2. Browse for suitable gene by description

3. Click ‘FASTA’ below title

4. Copy gene sequence

Obtaining regulatory parts (promoter as example)

iGEM part catalog (http://parts.igem.org/Catalog);

2. Browse the parts by type. (Take promoter as example) Click on promoter;

3. Enter promoter catalog;

4. Select desired function of promoter (Take constitutive as example);

5. Select your desired promoter. Check number of uses. The more the better. (J23100 as example);

6. Click part name x2;

7. Click ‘get part sequence’ at the top right hand corner of “Sequence and Features” box

8. Copy part sequence

Ordering synthesis parts (IDT)

It is highly recommended to obtain the parts from IDT or other DNA synthesizing company as the part distribution kit from iGEM is very unreliable. It is also much more convenient to just order the parts rather than using various assembly methods to assemble the plasmid. Note IDT does not allow any oligo orders exceeding 3000 bp. Please check the size of the plasmid that you wish to order by adding the size of your part and the pSB1C3 backbone together.

If the size of the plasmid (pSB1C3 vector+prefix+part+suffix) is smaller than 3000 bp, you should order it via the gBlock Gene fragment service. Please order it with the pSB1C3 vector and iGEM standard prefix and suffix unless otherwise specified.the steps are listed as below

https://sg.idtdna.com/site/Order/gblockentry
Sign in

3. Format sequence (pSB1C3 vector+prefix+part+suffix) https://ucckeigem.org/tools/

4. Copy and paste the sequence into the sequence box

5. Click test complexity

6. If the box below ‘test complexity’ shows red, optimize codon using http://sg.idtdna.com/CodonOpt. Repeat steps 3-6 until the box shows green. Even if the box is green, there may still be minor complexities. Read through the box and take your own risk

7. Click add to order (do not click 5' Phosphorylation option)

8. A biohazard disclosure pops up. Choose ‘no’ for every choice.

9. Let shek sign the signature box. Check the ‘I have read and accept …’

10. Click add to cart. You may check your order afterwards.

If the size of the plasmid (pSB1C3 vector+prefix+part+suffix) is larger than 3000 bp, you should order it via the Custom Gene service. However, as this service will come with an IDT Best-Fit vector, please do not order with the pSB1C3 vector. You are only required to input the part sequence and the igem prefix and suffix.

Common tools

NEBcutter

NEBcutter is an online tool developed by New England Biolabs. Its primary usage is to search for restriction sites in the provided DNA sequence. It is often used for two main purposes:

Check if there are more than one restriction sites for one restriction enzyme. This check is crucial as more than one restriction site for the same restriction enzyme could result in restriction digestion at unwanted locations, leading to an error.
Check if the designed restriction sites exists. If those restriction sites are somehow removed, the restriction enzyme wouldn’t work so the whole transformation process will fail.

The NEBcutter V2.0 can be found here: http://nc2.neb.com/NEBcutter2. Following is a step-by-step guide to use the tool for the above mentioned purposes:

1. Paste the DNA sequence in the text area and click “Submit”;

2. Click “Custom digest” under “Main options”;

3. Pick the specified restriction enzymes chosen to cut the sequence by checking the checkbox next to the name of the enzyme;

4. Click “Digest” at the bottom of the screen;

5. You should see the graph indicating the restriction sites of those selected restriction enzyme.

Codon Optimization Tool

Codon optimization tool is an online tool developed by IDT, a biotechnology company. It helps the user to optimize the sequence by replacing some codons using DNA’s degenerate property. This helps increase the protein expression and avoid restriction sites in the coding sequence.

The tool can be found here: http://sg.idtdna.com/CodonOpt. Following is a step-by-step guide to using the tool:

1. Choose the organism. This affects the optimization result as every organism’s protein expression process may vary a bit;

2. Paste the designed sequence into the textarea;

3. Click “Optimize”.

ORF Finder

An ORF Finder allows you to find all possible Open reading frame (ORF) within a given sequence. An ORF is part of a reading frame that has the ability to be translated, which spans from the start codon (ATG) to a stop codon (UAA, UAG or UGA). By finding the ORF, the sequence to be expressed can be predicted.

Link: https://www.ncbi.nlm.nih.gov/orffinder/ (NCBI)

1. Enter the sequence in the textbox.

2. Enter the bp of sequence that you wished to analyze. If you wish to analyze the entire sequence, just leave it blank.

3. Choose the Minimal ORF length (the minimum length of each ORF that is being searched) please choose the 30bp

4. Choose the origin of the sequence. You should check with this table to see which one category the gene originated from (link: https://www.ncbi...tgencodes)

5. For the start codon of the ORF, please choose ATG (the first option)

6. For the nested ORFs, please do not tick the box

7. Click submit

8. Results will be shown in the graph and the table.

(SAMPLE SEQUENCE)

BLAST

The Basic Local Alignment Search Tool (BLAST) is a powerful tool in comparing the query sequence(primary sequences eg.DNA and Amino acid sequence) to the NCBI database of sequence and identifies origin of the sequence according to its resemblance to the database. It provides a rapid way with an acceptable degree of accuracy to align sequences and identifies it, when comparing to other similar programs. It also helps the user to identify the originating organism of the sample, and thus helps with determining the phylogenetic relationship of 2 organisms.Link:https://blast.ncbi.nlm.nih.gov/Blast.cgi

There are various functions in the BLAST to allow for aligning different type of primary sequence(DNA or Amino acids). The most common types to be used is Nucleotide-nucleotide BLAST (blastn) and Protein-protein BLAST (blastp), which aligns DNA to DNA sequences and Amino Acid to Amino Acid sequence respectively. If you want to know more about BLAST, you can read this document for a more in depth guide on how to use BLAST (Link:ftp.ncbi.nlm.nih.gov/pub/factsheets/HowTo_BLASTGuide.pdf)

SnapGene

Snap gene is a collection of everyday molecular biology tools and viewers that is extremely useful to plan, visualize, and document on various cloning and PCR manipulation. One of the most used free function provided by snapgene is the snapgene viewer, which helps visualize, create, annotate, and share richly annotated DNA sequence files up to 1 Gb in length. It can be download for free straight from their website at:

http://www.snapgene.com/products/snapgene_viewer/

After you downloaded it, you can input any linearized or circular DNA sequence to it, and all possible restriction enzyme sites, possible genes and primers site will be identified. Editing and viewing the bp by bp is also possible with this application. Even translation ORF can be obtain through this viewer, making it a great software for handling routine molecular biology work.

UCCKE iGEM Tools

UCCKE iGEM Tools is an online tool developed by UCCKE Biology Research Team. The tool can be accessed here: https://ucckeigem.org/tools/. The current version provides 3 main functions, which are sequence formatting, comparing and analyzing.

The “Sequence formatting tool” will modify the sequence to a specific format: all caps, no space, no new lines etc.

The “Sequence comparing tool” will compare two user-inputted sequences and output a color-coded changes summary.

The “Sequence analyzing tool” will count the number of different nucleotides with some other analyzing functions.

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